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I. INTRODUCTION
Copper and zinc metabolism became easier to study after science provided
two basic research tools: isotopic tracers and atomic absorption spectropho-
tometry. The recent developments in cell and molecular biology have yielded
an additional array of techniques that have allowed the development of
exciting new insights into the absorption, metabolism, and function of these
trace elements.
Interest in these nutrients has grown dramatically in the last decade.
There has been an increased awareness that the levels of dietary intake of
copper and zinc may be marginal for patients with particular diseases or
for entire population groups. On the other end of the spectrum, pharma-
cological doses of these nutrients have been reported to have therapeutic
properties for specific diseases. In the past the total copper or zinc content
of a tissue was used to make judgments about biochemical changes and
metabolic fluxes of these metals. However, little was known about the bio-
logical environment around the metal atom or what influences its distribution.
Recently the perspective has changed dramatically. These metals were shown
to be interactive with intracellular components and influenced by the en-
docrine system in the same manner as the metabolism of the major nutrients.
Two examples of the biological interactivity of trace elements are the
regulation of a gene promoter by copper and zinc and the definitive endocrine
control of cellular copper and zinc metabolism.
This review integrates some of the widely held classic views of copper
and zinc metabolism with more recent discoveries at the cellular and molecular
level. It also relates these metabolic events to the biological functions of
copper and zinc. These newer concepts will improve our understanding of
the role these nutrients play in normal physiological mechanisms, host de-
fense, and other processes.
The basic aspects of copper and zinc absorption metabolism are shown
in Figures 1 and 2. To review the absorption mechanisms for copper and
Liver
FIG. 1. Basic aspects of mammalian copper metabolism. Dietary copper is absorbed from
stomach and small intestine. In human adults the RDA is -2.5 mg/day. Some copper in intestinal
contents is biliary and serves as the major excretory route. Normally only small quantities of
copper are lost in urine, but this increases during aminoaciduria. Copper is transported in portal
plasma, bound principally to albumin and possibly as amino acid complexes. Hepatic uptake
occurs via a saturable transport process. Systemic transport of copper from liver is primarily
as ceruloplasmin, which appears to donate copper to tissues. Circulating level of ceruloplasmin
increases in response to various stresses and disease-related processes.
240 ROBERT J. COUSINS
Liver/--)
In
FIG. 2. Basic aspects of mammalian zinc metabolism. Absorption of dietary zinc occurs
from the small intestine. In human adults the RDA is 15 m&day, of which 43 mg is actually
absorbed. Endogenous zinc is secreted into the intestine from pancreatic and biliary secretions
as well as from serosal-to-lumen zinc flux by intestinal cells. Zinc is not excreted in urine in
appreciable amounts except during concomitant excessive nitrogen excretion. Regulation of zinc
absorption occurs at cellular levels and through endogenous secretion. Absorbed zinc is transported
in portal plasma bound to albumin. Hepatic uptake occurs via a saturable energy-dependent
process and accounts for major initial accumulation of newly absorbed zinc. On the whole,
muscle and bone represent the largest pools. The latter probably is only returned to plasma
when bone is mobilized to maintain calcium homeostasis. Marked increases in muscle catabolism
may favor urinary zinc losses. Acute responses to physical stress and infection involve depression
in plasma zinc, uptake of zinc by the liver, and concomitant redistribution within hepatocytes.
It is possible that. both copper and zinc are transported across the brush
border surface of the small intestine bound to one or more absorbable ligands.
The chemical nature of these ligands has not been elucidated, although this
area has been enthusiastically pursued. Also, the kinetics of ion uptake may
argue against the concept of ion-ligand cotransport. Even if free copper or
zinc is transported, specific ligands may present these metals to the brush
Lumen Plasma
Albumin
Ku
Dietary
8 CU
Albumin
Endogenous \
I
cr
cu
Zn
cu
Zn
cu 1 Zn
Dietary
0l -.- - . . . . . . -. t
C nrlnntannrlc - \ PO01 / 0000 \
Albumin
\Zn
Functions Albumin
L
I
FIG. 3. Schematic representation of intestinal mucosal cell and probable sites of dietary copper and zinc interaction. Lumenal copper of dietary E
and/or endogenous origin is transported across brush border membrane surface (A). Transport of endogenous and/or dietary zinc is similarly z
shown in B. Potential interaction between high lumenal concentrations of copper and zinc for a common transport system, endogenous binding
factors, or receptors is shown as C. Once transported, these nutrient metals interact with intracellular pools. Copper and zinc are transferred from
enterocytes bound to albumin, probably at different sites on the molecule. If intracellular concentrations of copper or zinc are very high, because
of high dietary levels or elevated blood levels due to parenteral administration of these nutrients, promoter for the metallothionein gene is activated,
and enhanced transcription of the gene occurs. Normally the promotor appears more sensitive to dietary zinc levels than to dietary copper levels.
As the enhanced level of metallothionein mRNA is translated, cytoplasmic level of thionein polypeptides (wcwy line) is increased, and they bind
available cellular copper and zinc. Because the binding affinity of this protein is greater for copper than for zinc, eventually more copper is bound,
which prevents copper transport across basolateral membrane to plasma, thus reducing absorption. When intracellular level of thionein is sufficiently
high, zinc absorption is also reduced. Copper and zinc may compete for transport across the basolateral membrane. Serosal-to-mucosal fluxes of
copper and zinc are also shown, because this may contribute to intestinal endogenous secretion of these metals.
242 ROBERTJXOUSiNS Volume 65
Copper Absorption
L-Amino acids TT 226
Nitrilotriacetate TT 217
D-Amino acids T 226
Citrate, phosphate, gluconate 369
Oxalate, EDTA 58,226
High dietary protein 94,113; 170, 267
0 431
Histidine 0 169, 259
Phytate 94
Ascorbic acid 54, 194, 391, 440
Thiomolybdate 88, 421
Fiber 1 108, 219
Bile 168
Zinc 1 143,144, 301
Zinc Absorption
EDTA TT 150, 296, 298, 299, 451
High dietary protein 170, 396
Lysine, cysteine, glycine 166
Low dietary iron 181
Lactose 152,163
Histidine 453
299
Pancreatic secretions 120
9
Prostaglandin E2 400
0 203, 389
Ascorbic acid 359
399
Citrate 203
166, 201, 299, 362
275, 375
Picolinate 121,122, 375
0 145, 203, 299
275
Glutathione 0 275, 299
High dietary calcium 2,193
0 391, 404
Fiber 219, 321
0 108, 396
Low dietary protein 121,441
Phytate 296, 298, 389, 396
Copper 0 2
301, 439
High dietary iron 398
Effects on uptake vs. actual absorption in various species not separated. T, Increase; 0, no
effect; 1, decrease.
April 1.985 COPPER AND ZINC METABOLISM 243
and gluconate complexes may be more easily absorbed than complexes formed
with other ligands (369). Similarly, CuCO:<, Cu(NO&, and Cu-oxalate or Cu-
ethylenediaminetetraacetic acid (EDTA) are better sources of available copper
than CuS04 (58). Dietary phytate (94), ascorbic acid (54, 194, 440), thiomo-
lybdate (88, 421), and fiber (219) appear to complex with copper and limit
its absorption. Dietary fructose may also limit copper absorption and decrease
copper status during a marginal copper intake (152). The chelator nitrilo-
triacetate enhances copper absorption (217), perhaps by forming a complex
that is transported intact across membranes. However, this area requires
further study, particularly at the membrane level.
Endogenous secretiois appear to influence copper absorption. Bile has
a negative influence on the reabsorption of secreted copper (169); pancreatic
protein has a similar effect (205). Glucocorticoids promote biliary copper
secretion, which indicates that hormonal status could alter absorption by
influencing this excretion mechanism (23, 271). Owen (312) reported that
surgery and anesthesia associated with biliary cannulation reduced the copper
concentration in bile. In contrast, Schneeman et al. (368) found a lo-fold
increase in biliary copper concentration after similar surgical and anesthe-
tization procedures. These conflicting results could be related to the diets or
to the feeding behavior during the postsurgical period. How these findings
might relate to possible changes in copper absorption after trauma and stress
remains to be established.
Copper-64-binding substances from human alimentary secretions have
been isolated (168). Although these were not characterized, biliary copper-
binding components from gallbladder bile were of high molecular weight,
whereas those from hepatic bile, saliva, gastric juice, and secretin-stimulated
duodenal aspirate were of low molecular weight. Ekpatic and gallbladder
bile both inhibited 4Cu absorption based on whole-body retention. Therefore
it was suggested that these endogenous binding cokponents may act as
regulators of absorption by influencing the amount of copper available for
uptake. In these experiments, 4Cu was added in vitro; therefore these copper-
binding components could be a reflection of Cu binding to previously un-
occupied sites.
An interaction between copper and zinc may occur within the intestinal
lumen. For example, perfusion of the isolated intestine with solutions of high
zinc content caused some reduction in copper uptake in the mucosal cells
(301). Similar findings were reported in some studies with an in vitro gut
sac technique (4:39), but not all (143). However, it is not clear whether excess
zinc competes for a specific copper receptor on the brush border membrane
or at intracellular sites or competes for absorbable binding ligands. In this
regard it is interesting that a reduced dietary zinc supply enhances uptake
and absorption of copper as well as zinc (370, 388). This suggests that a
common site is involved in the uptake of these metals at the brush border
membrane, and/or the transfer of intracellular copper to the plasma is af-
fected.
Clearly, further study is needed to define the mechanism for copper
uptake by the brush border membrane.
absorption (441). This relationship may be coupled to the extent that the
proteins are degraded and/or to how tenaciously they bind zinc. Giroux and
Prakash (166) have shown that some zinc-amino acid ligand mixtures stim-
ulate absorption (e.g., lysine, cysteine, glycine) when absorption is measured
as the increase in serum zinc 3 h after oral intubation. Histidine has been
shown to increase zinc absorption in some studies (420,453), but not all (166,
299). Glutamic acid has been suggested as an important ligand for absorption
(261, 264). Heth and Hoekstra (193) demonstrated that high dietary calcium
in practical rations decreases zinc absorption, an observation that has con-
siderable practical application in the swine production industry. In contrast,
zinc deficiency did not influence calcium absorption by the perfused rat in-
testine (388). Dietary calcium did not appear to have a pronounced influence
on zinc absorption in studies with humans (391, 404).
Considerable controversy has developed over the reasons for the greater
bioavailability of zinc from human milk compared with cow milk. The ther-
apeutic valueof both zinc and human milk in treatment of acrodermatitis
enteropathica has spawned this interest (290, 293). This rare, autosomally
inherited human disease (293) is characterized by diminished zinc absorption
(245). It is possible that human milk contains a zinc chelate that is taken
up by the intestinal mucosa more effectively than zinc from milk without
this chelator. Citric acid and picolinic acid have been proposed as the agents
involved (123, 251); however, convincing evidence that these substances have
absorption-promoting properties has not been developed. When tested in the
isolated perfused rat intestine system, these substances did not increase zinc
uptake or portal transfer (299). Both citrate and picolinate significantly de-
creased the zinc uptake kinetics by intestinal BBMV (275). Picolinate had
no effect in rats (203) or calves with Adema disease (145). In contrast, in
other studies with rats, picolinate was found eflective in increasing zinc
absorption in a variety of dietary regimens in which absorption was decreased
(121,122). Compared with a number of potential ligands, 2-picolinate markedly
improved Zn uptake by everted intestinal sacs, whereas citrate inhibited
uptake (375). The 4-isomer of picolinate had the opposite effect. The Z-pi-
colinate-zinc complex was excreted to a greater extent by intact rats (375).
In one study, citrate slightly increased zinc absorption in intact rats (203),
whereas in others it had no effect (166, 201). Similarly in humans, citrate
did not increase zinc absorption from cow milk or infant formulas (362).
Zinc citrate has a negative charge at physiological pH, which could impede
zinc absorption (264). However, a similar situation exists for other binding
ligands with carboxyl groups, and some of these promote absorption, e.g.,
EDTA (150, 298, 299, 420).
An alternative explanation for the therapeutic value of human milk
compared with cow milk in acrodermatitis enteropathica is based on its
markedly different chemical composition (80). Low-molecular-weight Corn-
pounds that can bind zinc were present in both cow milk and human milk,
but the latter contained much less protein. Moreover the nature of the proteins
248
and other constituents (e.g., lactose and phosphorus content) in these milks
varies widely (206, 246, 270). The major zinc-binding protein in human milk
has been reported to be &her lactoferrin (27) or albumin (246), whereas
casein is the major zinc-binding protein in cow milk (206, 270). These milks
also differ markedly in free amino acids (160). Based on these differences,
the greater zinc bioavailabilitv in human milk mav be related partlv to
digestibility and binding affinitv of the constituent I;roteins. This proI~osa1
has received experimental s&ort (27, 246). The hioavailabilitv of iron in
human milk was shown to be greater than that in cow milk, and differences
in composition rather than the presence of specific chelators were used to
explain the difference (270).
It has been proposed that, the lipid fraction of milk is an important
determinant of zinc absorption, particularly during the neonatal period. Es-
sential fatty acids, particularly metaholites of linolenic acid [y-linolenic acid,
dihomo-y-linolenic acid, and prostaglandin El (PGE,)], may stimulate zinc
absorption and account for the relatively higher bioavailability of zinc from
human milk (86). This proposal is based principally on Zn-absorption mea-
surements wit,h neonatal rat,s, where the individual essential fatty acids were
injectsed intragastrically with 65Zn. Indomethacin inhibited this process, sug-
gest,ing that prostaglandin svnthesis was involved. Prostaglandins El and
Ez had no effect, however. Previouslv, SonK and Adham (400, 401) proposed
that, PGE2 mav be an endogenous intraluminal chelator of zinc that was
related to the mechanism of zinc absorption. Other investigators have not
confirmed an efYect of prostaglandins on the absorption of zinc (201, 389).
Secretion of zinc into the intestine occurs via pancreatic and biliarv
secretions and probably through cellular flux in the serosal-to-mucosal di-
rection. Endogenous zinc excretion seems to be significant in homeostasis at
zinc intakes above the dietary requirement (229, 4%). Ilowever, endogenous
secretions could contain factors that influence absorption. Early experiments
with Zn established that endogenous zinc was secreted into the int.estine
via pancreatic and biliary secretions (15, 26, 279, 381). There is considerable
variation among species, however. Sullivan et al. (418) found that t,he zinc
content of pancreatic secretions of zinc-deficient pigs was 25% of the normal
amount, alt,hough hiliary zinc output was not changed. The contribution of
biliarv excretion to endogenous zinc output appears to be low in pigs and
dogs (26, 324). Zinc secreted from the pancreas is primarily protein bound.
The protein and zinc contents of rat pancreatic juice are closelv correlated
(368). Pekas (325) observed that acetone precipitated nearlv di of the zinc
in porcine pancreatic juice. Also, Zn and [ %]histidine simultaneouslv were
incorporated in to pancreatic proteins and secreted. In comparison, when Zn
was added in vitro to cytosol from rat or canine pancreatic secretions, gel-
filtration-chromatographv elution profiles suggested that binding was to spe-
cies of ~1,500 daltons (120). Cousins et al. (81) have cautioned against this
latter approach, because anomalous results can occur with in vitro addition
of Zn if stoichiometric exchange does not occur uniformly with all ligands.
April 1985 COPPER AND ZINC METABOLISM 249
This situation may occur because only available sites may actually bind
exogenously added 5Zn, and these may have little biological relevance. The
findings of Lonnerdal et al. (250) on the distribution of zinc in bile and
pancreatic secretions agree with this interpretation. These authors observed
that when the actual zinc content was measured, rather than 65Zn binding,
zinc was shown to be bound to high-molecular-weight components in pan-
creatic secretions of rats. They found that biliary zinc was associated with
low-molecular-weight moieties, which agrees with the observation that glu-
tathione binds appreciable amounts of zinc in rat bile (3). Glutathione-de-
pleting agents lowered the biliary secretion of probable zinc-glutathione
complexes. A positive effect of glutathione on zinc uptake by BBMV or in-
testinal absorption has not been observed, however (275, 299). Zinc concen-
tration in rat bile is increased markedly for a few days after surgery (368).
In contrast to the focus given to bile and pancreatic secretions, Antonson et
al. (9) found that bile and pancreatic duct obstruction actually decreased
zinc absorption in rats. One study showed that pancreatic secretions are not
essential for adequate zinc absorption in human subjects (446), but another
study with humans suggested that normal pancreatic function was necessary
to absorb large doses of zinc (31).
Numerous small zinc-binding ligands from intestine have been postulated
to have specific roles in zinc absorption. Some of these putative factors were
reported to be of pancreatic origin; others are of undefined origin. The basis
for most of these relationships with zinc stems from the observation that
65Zn added to intestinal secretions or preparations frequently migrates on
gel-filtration chromatography as low-molecular-weight species (120). Amino
acids, a peptide, PGE2, and picolinic acid have been proposed as the binding
components. The interpretation of some studies is clouded because the evidence
is based on in vitro addition of Zn, which may not provide a representative
view of functional binding ligands, because binding may be nonspecific. More-
over, intestinal preparations from rats appear to be subject to proteolysis
in a way that alters the binding of zinc to various components (81,249). This
area has been extensively reviewed previously (351, 396).
Serosal-to-mucosal zinc flux through enterocytes may also account for
a portion of the zinc secreted into the intestine. Restriction of bile and
pancreatic secretions entering pig intestines does not prevent the secretion
of endogenous zinc via the feces, suggesting that secretion occurs via a serosal-
to-mucosal flux mechanism in intestinal cells (324). In chicks, fasting increases
the intestinal zinc content, mobilized from endogenous sources, without in-
fluencing absorption (215). Injections of EDTA reduced zinc secretion by rat
intestine (239). Methfessel and Spencer (280) found that rats given a 6Zn
dose intravenously secreted radioactivity rapidly and uniformly throughout
the small intestine. Davies, and Nightingale (92) obtained evidence for in-
testinal zinc secretion with Chelex resin-filled loops of rat intestine to measure
luminal appearance of previously taken up 65Zn. Similarly, Kowarski et al.
(235) observed an Na+-dependent serosal-to-mucosal transfer of 5Zn in everted
segments of rat intestine. Zinc-65 flux was greater in the jejunum and ileum
than in the duodenum, and active transport was shown in the serosal-to-
mucosal direction (toward secretion). Smith et al. (389) observed a higher
transfer rate of Zn from the portal perfusate during in vitro perfusion of
intestines from zinc-injected rats. IJsing the same system, Steel and Cousins
(412) did not observe significant differences in vascular-to-lumen zinc flux
with intestines from either zinc-adequate or zinc-deficient rats. Considerable
amounts of zinc from the vascular bed entered the intestinal cells, but little
was found in the perfused intestinal lumen. Ghishan et al. (162) measured
mucosal-to-lumen zinc flux (secretion) by perfusing rat intestines with so-
lutions of varying osmolality. Secretion was found to be greater in weanlings
than in suckling or adolescent rats. Davies (91) estimated that tl7$ of Zn
taken up by the int,estine is returned directly to the lumen. This flux was
greatest in ileal and jejunal segments. Paneth cells respond to changes in
intestinal zinc content,and Elmes (111) has suggested this may be part of
an excretory pathway for this metal. Glucocorticoids have been shown to
enhance Zn uptake and possibly the return of in to the lumen in recir-
culating perfusion experiments kith rat intestine (28).
beling experiments also indicated that this intestinal protein was inducible
by zinc administration (347). Inducibility was confirmed by feeding experi-
ments (t350) and injection studies (310). Little metallothionein was found in
intestines of zinc-depleted rats, whereas dietary repletion with zinc increased
metallothionein in a dose-dependent fashion (350). There is evidence that
high dietary copper will also induce this intestinal protein (178).
The inducible nature of intestinal metallothionein led to the suggestion
that the protein is an integral regulatory component for zinc absorption (75,
348, 349, 352). Although the responsiveness of biosynthesis to zinc is firmly
established, the role this protein has in the absorption process at normal
intake levels is not clear. Pulse-labeling studies and experiments in which
transcription was inhibited with actinomycin D demonstrated that metal-
lothionein gene expression in the rat intestine was increased in response to
zinc (347, 348, 352). Both metallothionein I and II genes were expressed in
the intestine in response to zinc administration (351). An acute increase in
metallothionein synthesis was correlated with a reduction in zinc absorption.
This relationship has been observed in whole-animal experiments and isolated
perfused intestines (276, t388). Binding of zinc, newly accumulated within
mucosal cells, to metallothionein was inversely related to the extent of ab-
sorption. Menard et al. (276) demonstrated that feeding a 150~ppm zinc diet
to rats, previously deprived of their zinc supply . for 4 days, increased levels
of intestinal metallothionein mRNA. The temporal relationship between me-
tallothionein gene expression, production of nascent metallothionein poly-
peptide chains that could bind intracellular zinc or copper, and an alteration
in absorption was convincing evidence that metallothionein biosynthesis will
lead to a reduction in zinc and copper absorption (75, 351). Zinc binding
occurred simultaneously with or shortly after release of the metallothionein
polypeptide chain from the polyribosomes (276). Therefore an alteration in
transcellular zinc flux produced by this mechanism would occur only during
or shortly after translation.
The metallothionein gene appears to be regulated in an inducible manner,
and an adequate stimulus must be provided for initiation of an elevated
transcription level. This seems to require either administration of fairly
large closes of zinc or copper or a sudden increase in the dietary supply of
these nutrients from a previously low intake level. This would be expected
if metallothionein was mainly a detoxifying agent. On the other hand, because
some metallothionein gene transcription is always occurring (276), this me-
tallothioncin is probably a regulator with a dampening effect on transmucosal
flux of zinc (and perhaps copper and cadmium). Clearly metallothionein could
have a dampening effect or could actually facilitate absorption, depending
on conditions of brush border uptake and efflux at the basolateral membrane
(75). Starcher et al. (410) have proposed a positive effect for metallothionein
induction on zinc absorption (actually measured as liver Zn uptake) in mice.
This, however, seems counter to what is known about the inverse relationship
between metallothionein gene expression and zinc-absorption rates in rats
252 ROBERT J. COUSINS Volume 65
(75, 276). In some studies Q-P may exchange with nonradioactive Zn2+,
which could be incorrectly interpreted as an increase in absorption and/or
uptake.
Intestinal metallothionein levels may not be altered to the same extent
in response to moderate dietary zinc or copper changes. In some cases these
dietary changes are not accompanied by simultaneous changes in apparent
absorption. This suggests that altered metallothionein biosynthesis may not
be a major factor in regulating the extent of absorption under normal dietary
conditions. Clear species differences exist, however, as shown by Flanagan
et al. (146) and Olafson (306), because there was no correlation between
metallothionein levels and zinc absorption in mice. However, the former
group observed with rats that zinc absorption was higher when metallothi-
onein levels were lower and vice versa. The homeostatic regulation of zinc
absorption clearly has other components. For example, when the dietary zinc
supply goes beyond the daily requirement, endogenous zinc excretion in-
creases. Weigand and Kirchgessner (455) have shown that over a course of
days after the dietary zinc intake level was increased, the intestinal secretion
mechanism for endogenous zinc in rats becomes the predominant regulating
component that determines net zinc absorption. The role that intestinal se-
cretion plays in zinc absorption in deficient or marginal intakes is not clear.
An active role for intestinal metallothionein as a negative regulator of
copper absorption was also proposed (125, 143, 144, 178). The mechanism
proposed was similar to that suggested for regulation of zinc absorption.
Saylor et al. (365) have reported- that changes in dietarv copper and zinc
levels fed to sheep did not affect metallothionein content of the intestine.
They proposed that the limited capacity of sheep to synthesize metallothionein
may account for the marked susceptibility of this species to copper toxicity.
Crane and Hunt (85) showed elevated intestinal metallothionein levels in
mottled mice. This may account for the high intestinal copper levels found
in these mutant mice. The relationship of this finding to human Menkes
disease, for which the mottled mutation is an animal model, is not clear.
Recent data from chromosomal localization studies have shown the metal-
lothionein I gene may be assigned to either chromosome 8 (82) or chromosome
16 (366) but not to the X chromosome, which is the site of the Menkes defect.
The mutual antagonism between copper and zinc absorption has focused
on the role that metallothionein might play in this interaction. The salient
features of this relationship are shown in Figure 3. Hall et al. (178) and
Fischer et al. (143) confirmed that high dietary levels of zinc depress copper
absorption. Also, high dietary copper can moderately depress zinc absorption
(178,301). Oestreicher and Cousins (301) found that more physiological levels
of dietary copper (1, 6, and 36 mg/kg) and dietary zinc (5,30, and 180 mg/
kg) had no mutually antagonistic effects. Wide differences in respective copper
and zinc concentrations in the lumen did have a mutually depressive effect,
however. The failure of copper to influence zinc absorption was shown in
experiments with perfused ileal sacs of rat intestine (2). These observations
April 1.985 COPPER AND ZINC METABOLISM 253
The binding ligand(s) that transports copper from the intestine to the
liver has not been extensively studied. Nevertheless it is generally assumed
that albumin carries out that function. Copper is loosely bound to albumin,
probably involving interaction with the terminal amino &oup, the imidazole
nitrogen of histidine at position 3, and other peptide bond nitrogens (188,
363). Van den I-lamer (444) suggested that the 5% of plasma copper that is
bound to albumin represents a stoichiometry of 1 atom copper per 1,000
molecules of albumin. However, perturbations in the concentrations of other
plasma constituents, e.g., a marked shift in plasma histidine content, could
alter this distribution. Hepatic uptake studies have shown that plasma copper
was accumulated whether it was presented to the liver as amino acid com-
plexes or as albumin-bound copper (118, 457). This indicates that albumin
would not have to be the plasma carrier from the intestine. Virtually all of
the newly absorbed copper is transported from the isolated, vascularly
perfused rat intestine as an albumin-bound complex (R. J. Cousins, unpub-
lished data).
Direct evidence has been obtained that albumin is the plasma carrier
for zinc as it leaves the intestine. Smith and co-workers (388, 390) used
affinity and ion-exchange chromatography plus polyacrylamide slab gel elec-
trophoresis to characterize zinc binding in portal plasma of intact rats and
in the portal effluent of isolated, vascularly perfused rat intestine. In these
in vivo versus in vitro approaches, virtually all of the absorbed zinc (Zn)
was bound to albumin, In comparison, newly absorbed Fe was shown to be
bound to transferrin with both experimental approaches. Evans and Winter
(128) previously suggested that portal zinc transport involved binding to
transferrin; however, Smith et al. (390) provided evidence that these con-
clusions were incorrect and were related to poor Zn recovery from the
chromatographic columns. Furthermore, by altering the composition of the
vascular perfusate to include albumin as an isosmotic replacement for all
plasma proteins, zinc absorption by the perfused intestine was doubled. Iron-
saturated plasma did not influence absorption, but elimination of all serum
proteins prevented absorption. These data suggest that the plasma albumin
concentration may influence the extent of zinc absorption. The observation
that isolated rat liver parenchymal cells will take up zinc from serum-free
media containing albumin indicates that albumin-bound zinc is available to
the liver (130). Collectively this evidence suggests that albumin is the principal
portal transport proteinfor these metals. As discussed in the next section,
copper and zinc do not appear to share common sites on the albumin molecule
April 1985 COPPER AND ZINC METABOLISM 255
(291, 326). A competition between copper and zinc at this point in the ab-
sorption process is thus unlikely (Fig. 3).
The nature of copper and zinc transfer across the basolateral membrane
of the intestine has not been studied directly. Recently, basolateral membrane
vesicles from rat intestine have been used to characterize the kinetics of zinc
transport across this membrane surface (300). The data suggest that a sat-
urable component with a K, of 63 PM and Jmax of 38 nmol Znmg- pro-
tein . min- is involved and may be regulated by an ATP-driven mechanism.
Aspects of copper and zinc absorption covered in section IIA-C have been
reviewed in considerable detail elsewhere (33, 76, 77, 98, 396).
A. Copper
Only -10-2(X% of the zinc in blood is found in the plasma (30, l317, 335,
373). The remainder is localized within erythrocytes in which carbonic an-
hydrase is the major binding site (199,437;. The erythrocyte membrane may
contain zinc. Bettger and ODell (24) have shown that zinc deficiency decreases
the ability of ervthrocytes to resist in vitro hemolysis. It was suggested that
zinc stabilizes the erythrocyte membrane. Despite this important function,
erythrocyte zinc uptake may be influenced by many fact,ors, as discussed by
Chesters and Will (67). Leukocytes also incorporate zinc, but there appears
to be no exchange with plasma zinc like that occurring in erythrocytes (97).
COPPER AND ZINC METABOLISM 257
Dreosti et al. (107) found that in contrast to plasma zinc levels, leukocyte c
zinc levels were not responsive to zinc depletion.
Plasma provides a metabolicallv active transport compartment for zinc,
and numerous factors influence the flux of zinc through it (17, 69, 107, 116,
241, 269, 321, 352). Most plasma zinc is associated with proteins, albumin
being the principal binding protein. This distribution varies among species,
but usually about two-thirds of the zinc in plasma is bound to albumin (30,
69, 164, 165, 335, 373). There is a good correlation between the changes in
albumin-bound zinc and plasma zinc concentration that occur during acute
and chronic disease (135,180). This pool of plasma zinc is frequently referred
to as loosely bound zinc. The ability of albumin to give up bound zinc may
be an essential feature of the transfer of zinc between plasma and tissues.
The association constant for the zinc-albumin complex is 10 (291). Serum
albumin levels may have some effect on serum zinc levels (179), although
this has not been a commonly observed relationship (16, 17). Smith et al.
(387) have described a family with hereditary hyperzincemia. Failla et al.
(134) demonstrated that this abnormality is the result of greater binding of
zinc to albumin in individuals with this defect. Albumin is also the principal
zinc-binding protein in human amniotic fluid (158).
Most of the nonalbumin protein-bound zinc in plasma was shown to be
associated with a2-macroglobulin (164, 165,317). Unlike albumin-bound zinc,
the zinc bound to cu2-macroglobulin has not been correlated with plasma zinc
levels (165). The zinc-cu,-macroglobulin association constant is >101 (291).
This tightly bound zinc has no known physiological function. During preg-
nancy the serum zinc concentration decreases proportionally in both the
albumin and cu,-macroglobulin factions (423). Morgan (289) described a his-
tidine-rich glycoprotein of low concentration in plasma but with marked
zinc-binding properties. In vitro studies with other plasma proteins showed
that this glycoprotein had the highest ability to bind zinc and could compete
successfully with these proteins for zinc. Its size, 58,000 daltons, indicates
that it would cochromatograph with albumin in most size-exclusion svstems.
The biological significance of this protein remains unknown. However, the
tight binding of zinc suggests that little dissociation would occur under phys-
iological conditions and that its ability to donate zinc to cells would be limited
unless endocytosis is involved.
Boyett and Sullivan (30) suggested that transferrin-bound zinc may con-
stitute a metabolically exchangeable zinc pool. Evans and Winter (128) pro-
posed that transferrin was the plasma protein responsible for portal zinc
transport. Three lines of evidence argue against this latter hypothesis. 1)
Charlwood (59) demonstrated that the equilibrium of albumin and transferrin
with zinc strongly favors binding to albumin. 2) Chesters and Will (68) dem-
onstrated in vitro that, at the concentrations these proteins are found in
plasma, more zinc is bound to albumin than to transferrin. In contrast,
Harris (186) has presented in vitro evidence suggesting that when serum
bicarbonate concentrations are considered, because thev. are required for zinc
binding to transferrin, this plasma protein mav have a higher aftinity for
zinc than does albumin. 3) Smith and co-workersused the isolated vasc;larly
perfused rat intestine to bind newly absorbed ;Zn to albumin, not to trans-
ferrin (388, 390). The latter studieswero conducted with vascular perfusates
containing bicarbonate (i.e., Krchs-Ringer solution) suggesting, at least at
the level of portal transport, that albumin is the more likely transport protein.
In experimental animals the zinc content of the plasma has been rapidly
reduced (within hours) when the dietary zinc supply was diminished (24;,
349, 350, 464). In humans the response tc; lower die&y zinc may occur more
slowly (3:3ci). A portion of the decrease, ;tt least in chicks, could be related
to changes in water metabolism (25). Plasma zinc concentrations increased
for a short time after large zinc doses or meals containing high zinc were
fed to humans or experimental animals (166, 269, 276, :321, z397). Sullivan et
al. (417) proposed the use of plasma response to oral zinc as a test of zinc
status in humans.
Chest.ers and Will (69) have evaluated plasma zinc flux (quantity of zinc
passing through plasma per unit time) in zinc-deficient and entlotoxin-treated
pigs. Flux was reduced to one-half in zinc-deficient, pigs compared with sup-
plemented controls. Endotoxin reduced zinc flux from plasma to tissues in
zinc-adequate but not zinc-deficient pigs. Note that infusion of zinc into some
of the endotoxin-treated pigs was lethal.
Weinberg (456) has proposed that the reduction in plasma zinc due to
infection is a protective function in a manner analogous to the role he assigned
for infection-induced hypoferremia. He used the term 31II trit iod in 11)21))ity
to describe this interpretation. Chvapil (70) has related hypozincemia to a
mechanism to activate phagocytic cells that are inhibited at normal plasma
zinc concentrations. The mechanisms for such an eflect may be partly hor-
monally controlled. For example, dexamethasone administration reduced the
plasma zinc content of rats (116). Interleukin 1 (IL-l; also called leukocvtic
endogenous mediator or endogenous pyrogen), turpentine, isopropanol, and
endotoxin had similar responses (17, 101, 117,211,323, :392,X93,426). Infusion
of ACTH also lowered the serum zinc concentration in humans. This reduction
may be limited to the albumin fraction (lt35). The plasma zinc-reducing action
of IL-l was diminished in streptozotocin-diabetic rats (X32). Cannon and
Kluger (53) found that human plasma removed after exercise and injected
intraperitoneally into rats elevated body temperature and depressed serum
zinc concentrations. An IL-l-like mediator was proposed as the agent re-
sponsible. Hypozincemia was shown to be concomitant with increased liver
zinc levels in burned rats, particularly those with complicating sepsis (3:33).
Also, serum zinc was decreased to :34X of normal in mice bearing plasma-
cytoma (141). This suggests that tumor growth may be associated with changes
in the metabolic flux of zinc, perhaps as a host-defense mechanism.
Acute starvation in humans appeared to increase plasma zinc (191).
Similar observations were made when zinc-deprived rats were subjected to
acute food restriction (269,349). The origin of the increased zinc is not known,
A /,r*il I!jS:i WPPER AND ZINC METAWLISM 259
but the effect may be a factor in the interpretation of data from zinc-deficiencv
studies when food restriction is also part of the experimental protocol. At-
tempts have been made to develop ways to maintain high plasma zinc levels
without continuous zinc administration. For example, systemic lupus ery-
thematosus in mice was markedly reduced when elevated serum zinc con-
centrations were maintained by continuous release of zinc from suhcuta-
neously administered suspensions of zinc pamoate (236 ). Similarly, Brewer
et al. (42) have shown that the plasma zinc concentration can be elevated
for long periods by administering zinc salts as oil suspensions that slowly
release zinc. This may have therapeutic application in human diseases, because
oral administration of zinc only yields a transient increase in plasma zinc
content (165, 321).
Only a small percentage of the total plasma zinc pool is bound to low-
molecular-weight ligands, usually histidine and cysteine. Henkin (188) has
proposed that the zinc bound to amino acids is in equilibrium with albumin,
but that bound to cu2-macroglobulin is not. Prasad and 0berleas (335) suggested
that the ultrafilterable (diffusible) amino acid-bound zinc plays an important
role in zinc transport. Histidine and cysteine are the aminoacids most fre-
quently implicated in this potential function. Yunice et al. (469) and Van Rij
et al. (446) have shown that infusion of amino acids increases urinary zinc
output. This suggests that elevated plasma amino acid levels may lead to a
shift in the plasma zinc equilibrium away from albumin binding. Important
changes in zinc excretion could occur as a result. Evidence that urinary zinc
losses follow that of nitrogen during muscle breakdown has been developed
(10,ll). Retention of plasma zinc with albumin prevents glomerular filtration
and thus precludes excessive urinary zinc losses that could occur with excessive
chelation of plasma zinc to amino acids. Because zinc excretion is increased
during stress and some diseases, the finding by Victery and associates (448)
that glucagon increased zinc filtration by the kidney is most relevant.
Vander Mallie and Garvey (445) developed a radioimmunoassay for me-
tallothionein. They observed that rabbit antisera raised against rat metal-
lothionein would cross-react with metallothioneins from various species, in-
cluding humans. Although not found in initial studies, further experiments
demonstrated the presence of metallothionein in rat serum at concentrations
of l-3 &ml (159). Using a sheep antibody of high titer, specific for metal-
lothionein I, Mehra and Bremner (273) found that administration of copper
or zinc increased plasma metallothionein I levels. Within 7 days after treat-
ment these levels were 4.1 and 1.6 r-q/ml, respectively. The physiological
significance of plasma metallothionein is not clear, but its concentration may
reflect metallothionein gene expression in tissues.
ucagon - - - - +
GI ucocorticoid - - -
: \ /
/ \ / J
I \ \ / Ceruloplasmln Apoceruloplasmin
. /
/ - .
/ / -- --
, --
Estrogen /
--- ------ -- -------
Testosterone
Bile Plasma
FIG. 4. Schematic diagram of liver parenchymal cell showing key components of hormonal regulation of copper metabolism and ceruloplasmln
synthesis and secretion. Copper uptake from the portal circulation may involve amino acid- and/or albumin-bound copper or free copper. Cellular
copper is distributed among various compartments, represented here as a copper pool. Epinephrine stimulation increases cellular copper accumulation
more than other hormones. Transcription of the ceruloplasmin gene could be regulated by glucocorticoids, CAMP, and/or copper. Epinephrine and
glucagon increase cellular CAMP levels. The number of exons (dark bars) and introns (light bars) for gene and formation of completed mRNA are
hypothetical. Synthesis of ceruloplasmin involves only membrane-bound polyribosomes. Secretion of ceruloplasmin as a single 1X2,000-dalton
polypeptide (including carbohydrate fragment) and concomitant change in plasma copper content is increased by glucocorticoids and to a lesser
extent by estrogen and testosterone. Interleukin 1 may increase plasma ceruloplasmin directly or indirectly via hormones. Glucocorticoids also
appear to increase biliary excretion of hepatic copper.
April 1985 COPPER AND ZINC METABOLISM 263
2. Zinc
Fluctuations in the dietary zinc supply cause small changes in the hepatic
concentrations of this metal (434). Basic aspects of zinc metabolism by he-
patocytes are shown in Figure 5. The fairly constant supply of intracellular
zinc is undoubtedly maintained through the concerted action of many hor-
monal inputs. There have been relatively few studies carried out to char-
acterize zinc uptake by liver. In early work in this area, Saltman and Boroughs
(361) demonstrated that zinc was accumulated by puffer fish liver slices
against a sevenfold concentration gradient. Low temperature slowed accu-
mulation as did incubation in the presence of dinitrophenol. Interestingly
iodoacetate and cyanide both enhanced accumulation. Initial binding or uptake
followed first-order kinetics with saturation occurring at >400 PM extra-
cellular zinc. In these experiments a passive mechanism of zinc accumulation
was proposed. More recent experiments by Failla and Cousins (130, 131),
with rat liver parenchymal cells maintained in primary monolayer cultures
as the model system, have presented a different view of hepatic zinc accu-
mulation. Zinc accumulation by these cells was temperature dependent, and
because inhibitors of oxidative metabolism (i.e., azide and cyanide) markedly
depressed zinc accumulation, energy dependence was suggested. Also, inhi-
bition by iodoacetate and N-ethylmaleimide indicates that reactive sulfhydryl
groups were required for one or more steps in the zinc-binding/translocation
process. Accumulation was found to be saturable at ~12 PM. This was in-
teresting because this value is remarkably similar to the normal plasma zinc
concentration (-15 PM). Incubation of the parenchymal cells with increasing
amounts of cadmium, up to a maximum of 10 PM, yielded a progressive
inhibition of zinc accumulation. These experiments were carried out in the
presence of insulin in serum-free medium containing bovine serum albumin.
Zinc accumulation by the primary cultures was characterized by two
distinct phases. The first was a fast-uptake phase, which probably represents
initial binding to specific sites on the plasma membrane surface. Because
most of the zinc in the portal plasma is bound to albumin, there could be a
zinc-albumin interaction with the plasma membrane at this point. The second
phase of accumulation was a linear function in which zinc was accrued at a
much slower rate (130). Subsequently, biphasic zinc uptake with freshly pre-
pared suspensions of rat hepatocytes was also demonstrated (408). Presence
of 10 PM cadmium produced a decrease only in the first phase of uptake. It
was concluded that only the first phase of accumulation was carrier-mediated,
and it was speculated that part of the uptake process for cadmium and zinc
occurred via a common pathway. Pattison and Cousins (319) compared the
65Zn-accumulation rate of parenchymalcells in monolayer culture to the total
CAMP/--------Y
Netallothionein Gene
I
Glucagon - - - --t \ -. .
Int erleu
/7
\
kin -7 Receptor\
\ \
\ / ,q 0 ,d I- \Nucleus
Free Polyribosomes
Albumm -Zn
Amino Acid%
April 1985 COPPER AND ZINC METABOLISM 267
lectively represented here as a labile zinc pool that accounts for accumulation, and a zinc-
metalloprotein pool that accounts for exchange. High intracellular levels of zinc, which can
follow increases in changes in plasma zinc concentration, activate the promotor for the me-
tallothionein gene. Expression of this gene seems to be more responsive to changes in dietary
zinc than in dietary copper. Glucocorticoids also activate transcription of this gene. Exons and
introns are approximately as established for the mouse metallothionein I gene (109). Nuclear
metallothionein has been detected (63), and this may serve a regulatory function related to gene
expression. Completed mRNA is 300-400 nucleotides and is translated as free polyribosomes.
Glucagon appears to exert its effect on metallothionein synthesis via both transcriptional and
translational regulation. Epinephrine may exert transcription of regulation as well. Glucagon
and epinephrine probably activate transcription via CAMP. Nascent metallothionein polypeptides
bind intracellular zinc and copper after transport into the cell. Zinc accumulation by liver cells
in response to endotoxin or interleukin 1 may act directly or through glucocorticoids and glucagon.
A variety of functions for metallothionein and thionein (apometallothionein) have been proposed.
These may be related to hormonal regulation of zinc metabolism. Intracellular copper also is
bound to metallothionein. These proteins differ in susceptibility to degradation in the order
thionein > zinc metallothionein > copper metallothionein. Lysosomal proteases appear to be
involved in degradation. Copper metallothionein resistance to proteolysis could partially explain
its accumulation in some genetic disorders, e.g., Wilsons disease. Plasma metallothionein levels
appear proportional to the extent that the gene is expressed.
268 ROBERT J. COUSINS Volume 65
B. Intracellular Compartmentalization
Both copper and zinc are distributed throughout the various subcellular
fractions of hepatocytes. Smeyers-Verbeke et al. (385) have analyzed the
nuclear, mitochondrial, rough and smooth microsomal, and supernatant frac-
tions of liver for protein, nitrogen, copper, and zinc. The average distribution
of copper in these cellular fractions was 27, 7, 7, 3, and 54%, respectively.
The average zinc distribution in these fractions was 23, 5, 12, 5, and 60%,
respectively. Clearly the supernatant fraction (in this case, 3 h at 100,000 9)
contained the highest percentage of both metals. This corresponded to ~36%
of the total liver protein and 40% of the total nitrogen content. Alfonzo and
Heaton (4) found that liver homogenates averaged 20.5 and 97.4 pg/g dry
matter for copper and zinc, respectively. The supernatant fraction represented
39.3 and 46.7% of the total copper and zinc content, respectively.
The supernatant fraction of liver appears to be metabolically active with
respect to copper and zinc. For example, copper deficiency in rats led to a
reduction in the copper content of all cellular fractions, but the supernatant
accounted for the most pronounced decrease (4, 285). The majority of 67Cu
from [67Cu]ceruloplasmin was shown to be taken up into the soluble fraction
of rat liver (258). However, copper-loading experiments generally produced
an increase in the copper content of the heavier subcellular particles (nuclear
and mitochondrial fractions) (126, 171). Lysosomes also accumulate appre-
ciable copper (447), which may result partly from binding of hepatic copper
April 1985 COPPER AND ZINC METABOLISM 269
1. Zinc
Efflux of copper and zinc from the liver undoubtedly depends on many
factors, specifically intracellular factors that might favor retention of these
April 1985 COPPER AND ZINC METABOLISM 271
metals within cells, and the availability of circulating ligands that could
transfer these metals from hepatocytes. Saltman and Boroughs (361) observed
such a rapid efflux of zinc from fish liver slices into Krebs-Ringer solution
that within 0.5-Z h, only a small portion of the accumulated zinc remained.
This observation is at variance with more recent data from studies with
isolated rat liver parenchymal cells in an albumin-containing medium, which
demonstrated that most of the newly acquired zinc was not readily available
for efflux. It appeared from these studies that efflux did occur, because within
lo-15 h the 65Zn uptake process appeared to plateau (130). Because cells at
this point were still able to take up 65Zn, it was concluded that this plateau
period was related to a steady state between cellular uptake and efflux. These
studies showed that induction of metallothionein synthesis, at least tem-
porarily, shifts this balance in the direction of accumulation. Pattison and
Cousins (319) have shown that dexamethasone increased the slow zinc ex-
change rate with a concomitant increase in the labile zinc pool of which
metallothionein appears to be a component. The estimate of total cellular
zinc turnover derived from these studies suggested total turnover occurred
in about 30 h at 16 FM extracellular zinc. In support of this estimate of rapid
turnover, Guzelian et al. (176) demonstrated that a functional zinc deficiency
occurred in primary cultures of rat hepatocyes when these cells were incubated
in a zinc-deficient medium. This suggests that a constant renewal exchange
process is necessary to maintain sufficient amounts of cellular zinc.
It is not clear to which plasma ligands zinc is transferred during efflux
from hepatocytes. Both albumin and amino acids are probably the ligands
involved. Trauma and other events that lead to muscle catabolism increased
plasma amino acids and subsequent amino acid excretion. A consequence of
this elevated excretion of amino acids is trace-element excretion (10, 11,140).
This could limit the supply of plasma zinc for the liver. Histidine was the
amino acid most frequently associated with urinary zinc excretion (188,469).
Further work is necessary to clearly evaluate this point. Moreover there is
substantial evidence that endogenous zinc is returned to the intestine as an
important part of intestinal zinc excretion and homeostasis (455). A small
portion of zinc excreted by this route may come from the liver via the bile.
2. Copper
A relatively low efflux rate of 64Cu was observed when rat liver paren-
chymal cells were incubated continuously with 64Cu for 6-12 h in albumin-
containing media (457). It was estimated that -15% of the intracellular
copper was lost in a l-h period, the majority being lost during the first 5-
to 15-min measurement period. This may represent exchange of copper with
a rapidly turning-over pool. In contrast to these studies, which were performed
with purified parenchymal cells that were maintained at 37C and exhibited
physiological responsiveness, Schmitt et al. (367) have shown that hepatocytes
maintained in suspension culture lose 35-40% of accumulated 64Cu during a
40-min preincubation period. These studies, however, were carried out at
272 ROBERT J. COUSINS b%wne 65
ZOC, and significant efflux was also evident at 4C, which questions phys-
iological significance. Efflux rates of 64Cu from rat liver slices reported by
Saltman et al. (360) were comparable to these. Using a liver perfusion system,
Owen and Hazelrig (315) found that a steady state between 64Cu influx and
efflux could be reached. Their efflux data were more suggestive of those
observed with isolated cultured liver cells (457).
Because glucagon increased copper uptake by cultured liver cells while
glucocorticoids increased copper secretion, the collective effect of these com-
pounds may be to enhance copper flux through the hepatocytes in the direction
of copper efflux as secreted ceruloplasmin. This may be important, because
it has been proposed that ceruloplasmin is the primary source of copper for
extrahepatic tissues (184). Glucocorticoids also increase biliary removal of
hepatic copper (23,271). Using a compartmental model for analysis, Vierling
et al. (449) showed a reduction of hepatic copper incorporation in patients
with primary biliary cirrhosis. Hepatocytes from brindled mice, animal mod-
els for Menkes disease, showed a defect in the overall copper-retention mech-
anism that appears traceable to an altered slow phase of efflux (90).
The pathogenesis of Wilsons disease includes an atypically high copper
accumulation by the liver and other tissues as well as decreased ceruloplasmin
levels and biliary copper excretion (114,171,443). Unfortunately the metabolic
basis of this disease is not known, but it could involve changes in the copper-
ceruloplasmin secretion process and/or changes in the hepatic synthesis of
metallothionein. The net result of either abnormality would be enhanced
hepatic copper accumulation and substantial copper binding to metallothi-
onein and intracellular localization. Protein synthesis may be a central factor
in regulating hepatic copper levels, because these levels increase during an
inhibition of either transcription or translation (172, 457).
VI. METALLOTHIONEIN
A. Properties
In the past decade metallothionein has been one of the most widely
studied metalloproteins. The reason for this, apart from its unique protein
chemistry, relates to its induction by heavy metals and hormones. Depending
on animal species and tissue from which it is isolated, this protein is found
to bind a variety of metals, particularly cadmium, copper, mercury, and zinc.
Equine kidney contains relatively large amounts of cadmium. In examining
the reason for the accumulation of this nonnutrient metal, a hitherto un-
discovered protein was isolated and characterized, which Kagi and Vallee
(210) termed metallothionein. The salient features of the protein are as follows.
I) It is a single polypeptide chain of 61 amino acids. 2) It has a metal-binding
capacity of between 5 and 7 g atoms/mol. 3) Twenty-five to 30% of the amino
acid residues are cysteine, and in the native protein there are no disulfide
bonds. (There is some evidence that under aerobic conditions and extensive
April 1985 COPPER AND ZINC METABOLISM 273
B. Synthesis
I. Regulation by metals
Inducer Ref.
Physiological
Development 7, 20, 38, 40, 220, 305, 467
Dietary zinc 36, 61, 144, 146, 178, 269, 276, 306, 349, 350
Infection 394
Starvation 35, 349
Stress 303, 305, 364
Experimental
Adjuvant arthritis 426
Alkylating agents 234
Cadmium 209
Carbon tetrachloride 51, 303
Copper 36, 37, 255, 278, 459, 465
Diabetes 133
Endotoxin 100, 117, 318
Epinephrine 31
Glucocorticoids 115, 116, 130, 131, 177, 213, 265, 342, 459
Glucagon 99, 115, 237
Interleukin lt 100, 102
Isopropanol 427
Retinoic acid 426
Turpentine 392, 393
Zinc 35, 37, 61, 109, 137, 178, 255, 302, 306, 310, 347, 348, 349, 350, 351,
352, 372, 378, 379, 380, 388, 406, 407, 410, 425, 430, 459
* General reference for induction by cadmium and metals other than copper or zinc.
t Also called leukocytic endogenous med iator or endogenous pyrogen.
April 1.tw.s COPPER AND ZINC METABOLISM 275
of gene expression. A major advance in this direction was the isolation and
characterization of the mouse metallothionein I gene by Durnam and co-
workers (110). To accomplish this a double-stranded cDNA was synthesized
from a mouse liver mRNA population that was enriched in metallothionein
mRNA. This DNA was subsequently inserted into the plasmid vector pBR322
and cloned in transformed Escherichia coli. A plasmid containing a 380 base-
pair fragment, which included the entire coding region, was used to identify
metallothionein I genomic clones prepared from a mouse embryo DNA library.
These clones were nick-translated with 32P-dNTPs and used as DNA probes
in hybridization experiments to quantitate metallothionein mRNA levels.
Specifically, cDNA was used to measure tissue mRNA levels, and genomic
DNA was used for in vitro transcription experiments (109). Their use has
provided more convincing evidence that administration of zinc, copper, or
cadmium, as well as dexamethasone, to intact animals will result in enhanced
transcription of the gene and increased production of metallothionein mRNA
(109, 177). Metallothionein gene sequences from humans (212, 214) and rats
(6) have also been cloned.
The responsiveness of the metallothionein gene to zinc was further es-
tablished by gene fusion experiments (316). The regulatory region on the 5
end of rat growth hormone was deleted, and the 5 region of the mouse
metallothionein I gene containing the promoter was added in its place. The
fusion gene was microinjected into fertilized mouse eggs. Many transgenic
offspring had higher-than-normal levels of serum growth hormone when
they were given extra zinc in the drinking water. This indicates that the
metallothionein promoter is sensitive to the level of zinc intake (as would
be expected from data derived from other experimental approaches). The
demonstration that the metallothionein promoter can be used to increase
expression of a fusion gene via an exogenous zinc supply suggests that this
approach could have value in activating fusion genes introduced into the
genome of plants or animals.
2. Dietary control
3. Hormonal regulation
of zinc may be transferred to the liver and other tissues; then metallothionein
synthesis is induced. How this event would relate to hypozincemia associated
with IL-1 administration is not clear (18). However, hypozincemia may be
a transient response to the initial induction of hepatic metallothionein syn-
thesis.
The influence of hormonal status and chronic hormonal imbalance on
copper and zinc metabolism and on hepatic and renal metallothionein has
been shown with the streptozotocin-induced diabetic rat. Failla and Kiser
(133) found that the concentration of both copper and zinc in these tissues
increased within a few days after treatment with this diabetogenic drug.
Changes in zinc distribution within the hepatic cytosol occurred prior to
changes in the absolute concentration of this metal in the tissue. It was
concluded that the plasma insulin-to-glucagon ratio and adrenal hormone
status, which are atypical in this diabetic animal model, were responsible
for the observed changes in copper and zinc metabolism. Because diabetes
involves hyperglucagonemia as well as hypoinsulinemia (435), these findings
agree with observations that glucagon has a pronounced regulatory effect
on hepatic metallothionein (99, 115). Furthermore the observation that the
tissue metallothionein content is altered in an animal model for a metabolic
disease, e.g., diabetes, points to the likelihood that tissue metallothionein
levels may be altered in similar conditions in humans. Plasma metallothionein
levels may be an early indicator of these disease processes.
Liver metallothionein increases dramatically during fetal growth. Such
observations h ave been made for various species, including humans . Porter
and Hills (331) proposed that neonatal liver mitochondriocupreine, a copper-
rich species, was a polymeric form of metallothionein. Bremner et al. (40)
found that most of the zinc and copper in the cytosol fraction of liver from
fetal sheep was bound to metallothionein. Ohtake et al. (305) postulated that
the function of metallothionein in neonatal metabolism was related to DNA
synthesis in liver. Bell (20) observed that maternal metallothionein induction
in rats was independent of fetal hepatic metallothionein. Kern et al. (220)
detected fetal metallothionein in rats as early as 14 days after conception
and observed that it increased steadily until term. Maternal levels were
relatively constant. The authors proposed that metallothionein synthesis
may signal the shift from proliferation to differentiation. Metallothionein
mRNA levels were increased in fetal mouse (309) and fetal rat tissues (7).
In the rat fetus these levels increased steadily to term, remained at that
level during the nursing period, and decreased thereafter. In contrast, the
mRNA level in maternal liver was highest immediately before parturition.
Levels decreased between days 17 and 20 of gestation and were correlated
to the increased maternal-to-fetal placental transfer of zinc obser ved during
that period (136). These changes in maternal mRNA were correlated to serum
progesterone levels that may suppress maternal metallothionein mRNA
through an inhibition of induction (7). It has not been established whether
fetal metallothionein increases because of elevated fetal zinc and/or copper
284 ROBERT J. COUSINS vohme 65
C. Degradation
D. Function
VII. CERULOPLASMIN
Holmberg and Laurel1 (196) named the blue protein from plasma ce-
rulorplasmin. Recent detailed reviews of the physical and chemical properties
of this cuz-globulin are available (155, 240). Only general properties of ce-
ruloplasmin are described here. Ceruloplasmin from human plasma is a gly-
coprotein of -132,000 daltons containing 6 copper atoms per molecule and
78% carbohydrate. Proteolytic cleavage of intact ceruloplasmin may occur
during preparatory steps unless precautions are taken (224,357). Three major
polypeptide fragments of 67,000, 50,000, and 19,000 daltons collectively con-
stitute the entire single polypeptide chain (428). These fragments may relate
to specific proteolysis-resistant domains within the molecule.
Frieden (155) has reviewed in depth the catalytic activity of ceruloplasmin
and has divided its potential functions into five categories: 1) oxidation of
Fe2+ as it is released from hepatocytes and converted to Fe3-transferrin in
the regulation of hepatic iron mobilization; 2) oxidase activity for aromatic
amines; 3) transport of copper to tissue sites; 4) serum antioxidation, in which
it acts as a scavenger of free radicals and superoxide ion; and 5) endogenous
modulation of the inflammatory response. The last three potential functions
are most relevant to the physiological regulation of hepatic copper and are
emphasized here. Function 5 is most likely related to functions 3 and 4,
because these are most closely linked to host-defense mechanisms.
Because ceruloplasmin represents 90-95% of the serum copper content,
it could be expected to be a primary source of copper for extrahepatic tissues.
For example, tissue cytochrome c oxidase activity in copper-deficient rats
was increased to a greater extent by ceruloplasmin copper than by copper
salts, copper histidine, or albumin complexes (200). Based on this evidence,
Frieden (155) proposed that ceruloplasmin Cu2+ is reduced at a cell membrane
receptor, and Cul+ is subsequently transferred to an unidentified intracellular
acceptor. Alternatively, intact ceruloplasmin may be taken up by endocytosis,
and Cu+ may then be released by proteolysis or a mechanism that subse-
quently recycles the protein to the plasma membrane for release. Owen (311)
made a similar proposal for the copper-donating potential for ceruloplasmin.
Linder and Moor (244) presented evidence that ceruloplasmin enters cells.
The net result of either route of copper entry into cells could be that Cul+
is oxidatively transferred to apoenzymes. Harris and co-workers (185, 416)
have presented evidence for a ceruloplasmin receptor in chick aorta and have
shown that the activation of aortic lysyl oxidase was correlated to cerulo-
plasmin levels (184). In vitro activation required oxygenation and protein
synthesis. Roles for ceruloplasmin in angiogenesis (344) and cell growth (263)
have been proposed. Weiner et al. (463) have reported improved performance
in successive generations of sheep when selection is based on high versus
low plasma copper. Presumably this is a genetic selection for high plasma
April 1985 COPPER AND ZINC METABOLISM 289
Inducer Ref.
Physiological
Cancer 72
Chronic inflammation 73, 96, 153, 282, 424
Development 57, 126, 327, 468, 470
Exercise 106, 182
Pregnancy 147
Experimental
ACTH 5, 154, 411
Copper 125, 243, 458, 462
Endotoxin 87, 117, 154, 355
Epinephrine 154, 281, 458, 459
Estrogen 72, 119, 147, 281, 330, 356
Glucocorticoids 5, 87, 411, 459
Inflammation
Adjuvant arthritis 426
Carrageenen 282, 283
Turpentine 281
Interleukin 1 452
Retinoic acid 426
Zinc 426
requirements of the fetus (190). Planas and Frieden (330) observed that
estrogens induce ferroxidase activity (presumably ceruloplasmin) in normal
and copper- or iron-deficient roosters. Earlier, estradiol was found to increase
serum ceruloplasmin in rats (281). In humans, elevated testosterone levels
were correlated with increased serum ceruloplasmin (208, 470).
Adrenal hormones have received particular attention regarding their
effect on ceruloplasmin levels in the blood. The regulation of ceruloplasmin
synthesis and secretion by glucocorticoids is similar to that documented for
other acute-phase proteins, e.g., fibrinogen (173). The early experiments
showing the effects of adrenalectomy on plasma copper and ceruloplasmin
have been reviewed (118). Adrenalectomy increased serum ceruloplasmin
levels as well as hepatic copper concentrations. Evans and Wiederanders
(127) found that corticosterone injections blocked this increase in adrenal-
ectomized rats. Adrenalectomy did not influence serum ceruloplasmin in the
experiments of Meyer et al. (281). Corticosteroids appeared to promote hepatic
copper excretion via bile (23,271). Adrenalectomy appears to have a cholestatic
effect, and the resulting increase in the intracellular copper may have marked
effects on ancillary processes, e.g., metallothionein induction. Epinephrine
was shown to increase serum copper and ceruloplasmin in both intact and
adrenalectomized rats (281). Vigorous physical exercise in humans also in-
creased serum ceruloplasmin (182), as does swimming in rats (106). The
exercise would be expected to increase plasma epinephrine levels and IL-l
production.
Interleukin 1 can increase serum ceruloplasmin levels (452). Cannon and
Kluger (53) have shown that an IL-l-like factor bound in human plasma
after exercise causes hypozincemia and hypoferremia. The increase in serum
ceruloplasmin after exercise is possibly related to IL-l release (106, 182).
Activation of macrophages to produce IL-l could in turn signal hepatic ce-
ruloplasmin synthesis. However, IL-l may act via a combination of hormonal
signals or in conjunction with them (332). The increase in muscle catabolism
associated with fever may be induced by IL-l, being mediated via PGE2 (14).
Unfortunately the effect of prostaglandins on ceruloplasmin synthesis has
not been investigated.
Ceruloplasmin induction by copper administration has yielded equivocal
results. Holtzman and Gaumnitz (197) did not observe an effect in copper-
deficient rats during a 2- to 8-h time course. In contrast, Linder et al. (243)
observed that copper administration increased ceruloplasmin in copper-de-
ficient rats but not those of adequate copper status. Milne et al. (286) reported
a similar induction of ceruloplasmin synthesis in copper-deficient rats. High
dietary levels of silver, cadmium, and zinc appeared to reduce ceruloplasmin
levels (462). Evans et al. (124) detected increased ceruloplasmin synthesis in
response to large doses of copper in experiments in which [14C]lysine incor-
poration into ceruloplasmin was measured by chromatography. Actinomycin
D blocked the induction. It was postulated that regulation by copper was at
the transcriptional level. Linder et al. (243) have suggested that the large
294 ROBERT J. COUSINS vi&wne 65
gested that when the extracellular copper content was sufficiently high, ce-
ruloplasmin gene expression may have been increased. This agrees with the
intact-animal data of Linder et al. (243), who demonstrated an inductive
action for exogenous copper when the dietary copper supply was diminished.
In this case, the sudden influx of copper associated with a large dose of
copper may be sufficient to bypass normal controls and activate the gene or
Fever
Gl ucogon ! Glucocorticoid
Fe
Plas mo Zn
Cu - Ceruloplasmin \
Toxins
Detoxified
Product
Tissue Cu Antioxidant
Fe II---- Fe IIf
FIG. 6. Hypothesis for integrated hormonal and mediator-controlled changes in hepatic
copper and zinc metabolism as related to host defense. Stress and trauma increase plasma
glucagon and glucocorticoids as well as interleukin 1, which in turn increase metallothionein
levels in liver cells and promote ceruloplasmin secretion. Increased metallothionein and zinc
redistribution in liver could relate to altered enzyme activity, membrane stabilization, and/or
detoxification mechanisms. Elevated plasma ceruloplasmin relates to increases in ferroxidase
activity, copper transport to tissues, and serum antioxidant properties, which may preclude
deleterious oxidative damage. Macrophages also synthesize metallothionein in response to glu-
cocorticoids (318). This change is correlated to the ability of the macrophage to combat the
inhibitory influence of endotoxin on phagocytosis. Integral role of interleukin 1 in these stress-
related phenomena and fever is indicated.
296 ROBERT J. COUSINS Vdume 65
VIII. CONCLUSION
Aside from the well-documented roles for copper and zinc in metal-
loenzyme activity, there have been few attempts to combine observations
such as those reviewed here into a unified hypothesis of a biological role.
The schematic diagram in Figure 6 shows how the findings on the hormonal
regulation of copper and zinc metabolism could relate to overall host-defense
processes. The function of metallothionein has been widely addressed and
may be a central component of homeostatic mechanisms. Fasting, trauma,
stress, and infection influence the cellular levels of this protein. The functions
of ceruloplasmin that could relate to defense mechanisms have been discussed
as well. The evidence for these potential roles is accumulating and will lead
to further experiments designed to answer specific questions.
In this review, I have discussed some of the more recent information
regarding the absorption, transport, and hepatic metabolism of copper and
zinc. There are many excellent monographs and reviews that address these
topics from various perspectives (2, 44, 48, 65, 129, 209, 295, 313, 314, 343,
403,433, 434).
The last decade has seen unprecedented research activity on these metals.
In an attempt at brevity, and at the same time with a desire to keep the
review contemporary, a large segment of classic observations had to be omit-
ted. However, the state of knowledge in this area is based on past decades
of research when the fundamental aspects of metabolism of these metals
developed. It is clear from the examples cited that the metabolism of these
nutrient metals respond to physiological stimuli at many levels of biological
organization and in ways we are only beginning to understand and appreciate.
I thank Ann C. Coutu for typing the manuscript, Walter Jones for drawing some of the
figures, and various colleagues who have critically evaluated the manuscript and made valuable
suggestions. Research from my laboratory discussed in this review was supported by National
Institutes of Health Grants AM-31127, AM-31651, and ES-03103. This review is Florida Agric.
Exp. Station Journal Ser. No. 5796.
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