Absorption, Transport, and Hepatic

Metabolism of Copper and Zinc: Special

Reference to Metallothionein
and Ceruloplasmin
ROBERT J. COUSINS

I. Introduction .......................................................... 238

II. Absorption of Copper and Zinc ........................................ 239
A. Cellular uptake from intestinal lumen .............................. 240
B. Intracellular compartmentalization within intestinal cells ........... 250
C. Transfer to portal circulation ...................................... 254
III. PlasmaTransport .................................................... 255
A. Copper ............................................................ 255
B. Zinc .............................................................. 256
IV. General Functions of Copper and Zinc ................................. 259
V. Hepatic Metabolism of Copper and Zinc ............................... 261
A. Uptake from plasma ............................................... 261
B. Intracellular compartmentalization ................................. 268
C. Copper and zinc efflux from hepatocytes ............................ 270
VI. Metallothionein ...................................................... 272
A. Properties ........................................................ 272
B. Synthesis ......................................................... 273
C. Degradation ....................................................... 284
D. Function .......................................................... 286
VII. Ceruloplasmin ........................................................ 288
A. Properties and functions ........................................... 288
B. Synthesis and secretion ............................................ 289
VIII. Conclusion ........................................................... 296

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

238 0031-9333/85 $1.50 Copyright @ 1985 the American Physiological Society

April 1x6 COPPER AND ZINC METABOLISM 239

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.

II. ABSORPTION OF COPPER AND ZINC

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.

zinc and to develop strategies to approach this area experimentally, it is

helpful to examine the distinct phases of absorption separately. This is not
always possible because most, studies have not been designed to evaluate
distinct steps of the processes involved. In this review the term tc~~~q~f~c~~~
is limited wherever possible to copper or zinc that has entered the portal
circulation after being transferred from the intestinal lumen and through
absorptive cells. Figure 3 is a schematic showing sites of control of copper
and zinc absorption. A partial list of factors that influence the absorption
of these nutrients is shown in Table 1; the list does not include physiological
status, which can also affect absorption.

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

TABLE 1. Eects of dietary constituents, metal-binding agents,

and endogenousfactors on copper and zinc absorption
Constituent Effect? Ref.

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

border membrane surface in a way that increases uptake. The respective

acidic and neutral environments of the stomach and small intestine and the
extent of digestion undoubtedly affect the interaction of these nutrient metals
with a variety of other dietary components in ways that influence absorb-
ability. Some nonabsorbable ligands may limit copper and/or zinc absorption
if the binding constants are sufficiently high to prevent either release of the
metal for transport as a free ion or rebinding of the metal to a more absorbable
binding ligand.
I. Copper

Many dietary factors influence the uptake of copper by the gastrointes-

tinal tract (Table 1). A portion of the dietary copper supply was shown to
be absorbed from the stomach (49,442). The acidic environment may promote
copper solubility and enhance its transport across the gastric mucosa (259).
Interaction between copper and other metals may also influence copper uptake.
This would most likely occur in the stomach because of the prevalence of
ionic copper in the g&x% secretions, where direct competition with other
metals could take place.
Copper transport across the brush border membrane of the small intestine
may be influenced by a variety of dietary components, including fiber, phytate,
ascorbic acid, thiomolybdate, and amino acids. The extent of exchange of
copper with intestinal binding ligands is not known, but competition for
various ligands could also be a factor that influences the extent of uptake.
The evidence that amino acids affect copper uptake from the intestine
has a stronger experimental basis than that for zinc. Kirchgessner and
Grassman (226) found that copper-amino acid complexes were absorbed to
a greater extent than CuS04. Specificity was suggested because complexes
with L-amino acid were more effective than those with D-isomers. In other
work, copper-histidine complexes did not improve copper absorption rates
(169, 259). High protein diets have generally been shown to exert a positive
influence on copper absorption (94,113,170,267), but not always (431). Protein
digestibility could have a marked effect on the extent to which copper-amino
acid and peptide chelates are formed. These chelates could enhance bio-
availability directly or lessen the binding of copper to other dietary com-
ponents that could affect bioavailabilitv. For example, the proteins in human
milk are more easily hydrolyzed by human neonates than the proteins in
cow milk. In human milk, 28 and 39% of the total copper has been reported
to be bound bv casein and albumin, respectively, whereas lower-molecular-
weight components bind 24% (246). However, an earlier report showed no
evidence of a low-molecular-weight copper complex (247). The lack of a ther-
apeutic value of human milk in treatment of Menkes disease was ascribed
to the absence of this factor. In cow milk, casein is the principal copper-
binding protein (246).
Complexes of copper not directly related to protein intake may also
influence copper uptake from the gastrointestinal tract. Citrate, phosphate,
244 ROBERT J. COUSINS Volu TTLV65

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.

Much more information is available on zinc uptake into the intestinal

mucosa than is available for copper. Dietary factors that influence zinc ab-
sorption are shown in Table 1, The general consensus that zinc absorption
is homeostatical~y controlled agrees with the original observations of Cotzias
and Papavasiliou (74); however, the exact mechanisms involved have not
been characterized. Unlike copper absorption, zinc absorption is believed to
be limited primarily to the small intestine. This is interesting because the
acidic conditions of-the stomach might, be expected to enhance zinc solubility
and provide an excellent environment, for uptake. In this regard the absorption
of zinc is more like that of calcium, which is limited to the small intestine.
The primarv site of uptake has not been established, but all regions of the
intestine may contribute to some extent (396).
Recent data suggest that zinc uptake across the brush bort-ler surface
may occur partly by a regulated carrier-mediated diffusion mechanism. The
velocity of zinc uptake by brush border membrane vesicles (BBMV) was
increased when they were prepared from zinc-deficient rats compared with
controls (5.4 vs. 12.0 pm01 . min? l mg -l protein), but the K, (0.4 mM) did
not change (274)- These findings suggest thf:re is a brush hort3er mcmbrantt
mechanism that responds homeosttttically to the dietary zinc supply. Trans-
port kinetics showed evidence of both passive (nonmediated) and saturable
(mediated) processes. With BBMV from zinc-depleted rats, a greater portion
of total transport was from the mediated (saturable) component. However,
in these experiments the nc~nmediatd component may represent zinc leakage
across a membrane damaged by high zinc concentration. Furthermore it was
observed that many brush border membrane proteins are influenced by nu-
tritional status, including zinc depletion (277). Sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) demonstrated that the abundance
of a membrane protein of -45,000 daltons was increased dramatically within
the first few days of zinc restriction in pair-fed rats compared with controls.
The protein al& was observed, albeit to a more limited extent, during total
food restriction. An alteration in a membrane zinc t.ransport protein(s) could
account for the differences in zinc uptake by BBMV mentioned above. This
possibilitv is being investigated. Such an induction couId explain the mech-
anism for the apparent increase in t/race-element absorption observed during
a fast (341) or during pregnancv and lact,ation (93). Numerous experiments
have described the biphasic na&re of zinc uptake 1~~ the intestine (245, 320,
359). Some phase of zinc uptake or tranq,ort ma&equire energy, but the
evidence for this is conflicting. In some stuciies inhiiiitors of enerm production
had no eflect (320, 359), whereas in other studies a clear effect was observed
(235, 371). This area has been reviewed in detail (396).
 Additional evidence shows that a diminished supply of dietary zinc may
increase zinc absorption at the uptake phase. Using different approaches,
Davies (91) and Smith and Cousins (388) observed two phases of zinc ab-
sorption as a function of the luminal zinc concentration. A fast mucosal
uptake phase across the brush border membrane was followed by a slower
phase that probablv involves transport of zinc across the basolateral mem-
brane. The fast phase of uptake, with increasing luminal zinc concentrations,
may represent saturability of binding sites on the brush border membrane.
At high zinc concentrations the membranes mav become leaky and allow
zinc to enter the cell and bind nonspecifically to cell proteins and other
ligands.
Jackson et al. (203) have also suggested that two mechanisms of zinc
absorption exist. One of these may be induced by low dietary zinc levels. The
finding that zinc absorption mav have a more pronounced mediated component,
in response to zinc depletion supports this hypothesis (274, 412). Moreover,
because altered brush border membrane transport mav account for a larger
segment of the zinc absorbed at lower luminal zinc concentrations in rats
fed zinc-deficient diets (412), the total amount of zinc absorbed when dietary
zinc is low may be close to that observed with higher dietary zinc intakes.
Davies and Williams (93) have reported that zinc absorption is greatly
increased in rats during the latter stages of pregnancy and during lactation.
The effect appears to be somewhat specific, because no change in lysine
absorption was observed. Similarly, Quarterman and Morrison (341) have
reported that the absorption of numerous heavy metals, including copper
and zinc, are increased during periods of food restriction.
The chemical form of transported zinc is not known. IJptake at the
membrane surface, perhaps via an unidentified receptor, may require bind-
ing of free ions or prior binding to specific compounds. This important question
has received vigorous attention, because anv factor that would enhance the
availability of zinc to receptors on the brush border surface might increase
bioavailability; however, the issue needs clarification. Zinc uptake has been
demonstrated with BBMV without added chelators (274,275), and zinc uptake
from ligated intestinal segments filled only with saline can be demonstrated
(91,235,442). However, transport as a comDlex is likelv in some cases. EDTA
has a high binding affinitv for zinc (260, 450), and it has been shown, at least
in presence of phvtate, to enhance zinc absorption in animals (150, 296, 359,
451). Suso and Edwards (419, 420) found that the EDTA-zinc complex may
traverse the intestine to the portal circulation intact. Oestreicher and Cousins
(299) found that when EDTA was introduced into the lumen perfusate, it
reduced the amount of zinc retained within mucosal cells of the isolated,
vascularly perfused rat intestine but significantly increased that transferred
to the portal circulation. This could be interpreted as an increase in zinc flux
through intestinal cells mediated by a cotransported chelator of high affinity.
High levels of dietary protein have frequently been reported to stimulate
zinc absorption (170, 396); conversely, protein restriction was shown to limit
 COPPER AND ZINC METABOLISM 247

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).

The intracellular distribution of intestinal copper and zinc has been

extensively studied. The basic concepts involve hypothetical pools for each
element and distribution of the metals to specific cellular sites for definitive
functions, e.g., zinc to membranes for stabilization and to metalloenzymes
and possibly to intracellular binding sites as part of regulatory or defense
mechanisms. Copper is probably directed to the site of copper metalloenzyme
synthesis. Copper and zinc are both toxic to cells in high concentrations.
Therefore, any aspect of intracellular binding that limits the availability of
metals for participation in deleterious reactions could be viewed as defen-
sive (98).
Most of the attention given to copper and zinc binding within intestinal
cells has focused on macromolecules, particularly metallothionein, in the
cytosol fraction (see Fig. 3). However, most of the copper and zinc in this
fraction is usually associated with higher-molecular-weight proteins. Starcher
(409) reported that Wu would bind principally to a lO,OOO-dalton soluble
intestinal protein when the isotope was orall; administered to chicks or
added in vitro to duodenal homogenates. On the basis of this molecular
weight and the sulfhydryl content, Evans et al. (125) proposed that this 64C~-
binding moiety was metallothionein. The basic characteristics of metallo-
thionein are discussed in section VIA. Richards and Cousins (351) purified
the protein to homogeneity . from intestines of zinc-injected rats and identified
it as metallothionein (351). Five criteria were used: amino acid composition,
sulfhydryl content, metal binding capacity, dimorphic behavior on diethyl-
aminoethyl (DEAE) chromatography, and apparent molecular weight. La-
 251

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

generally support earlier data on this relationship developed by Van Campen

(439) but suggest that wide dietary imbalances of copper and zinc are necessary
to observe an effect.
Metal1 0th ionein appeared to be the locus that accounted at least partly
for the copper-zinc interaction (144, 348). The responsiveness of the metal-
lothionein gene in the intestine to regulation by copper has not been well
studied. However, recent studies showed that intestinal metallothionein in-
duction is more responsive to dietary zinc than to copper (301). Assuming
that either metal can induce the protein, the higher binding affinity of me-
tallothionein for copper relative to zinc could explain why with moderate
mucosal metallothionein levels a reduction of copper absorption may occur
before a reduction in zinc absorption. High levels of intestinal metallothionein
may be required to decrease zinc absorption. Further support for this concept
is the finding that the marked association of copper with metallothionein in
intestines from rats fed high zinc diets occurs during the absorption process,
whereas the basal level of intestinal copper bound to metallothionein is very
low (301). This suggests that one phase of the copper-zinc interaction occurs
as these metals traverse the intestinal cell for transfer to plasma.
The copper-zinc interaction may have clinical importance when phar-
macological doses of these metals are given. Both metals have been suggested
for treatment of rheumatoid arthritis and other diseases. Therefore the con-
sumption of megadoses of either of these nutrients will probably make the
interaction a practical concern. For example, Prasad et al. (334) observed
hypocupremia induced by zinc therapy. Nevertheless the copper-zinc inter-
action may have some selective beneficial effects. Recently, oral zinc therapy
was shown to have therapeutic value in Wilsons disease, probably through
a limitation of copper absorption (43, 198). To maximize the therapeutic
regimen, Brewer et al. (43) administered zinc five times per day. These ob-
servations may relate to intestinal metallothionein induction. If the same
basic time tour se of induction foun d in rats (276) applied to h umans, this
regim en would maxi mize intestinal metallothionei n synthesis.
Some evidence suggests that zinc absorption is altered by hormonal
status. Absorption of both copper and zinc was increased in streptozotocin-
diabetic rats without changes in intestinal metallothionein (84). Polyphagia
or increased uptake of both copper and zinc at the brush border membrane
may be the cause of this effect. However, with the same model, perfusion
studies showed that lumen-to-mucosal and mucosal-to-lumen zinc fluxes were
decreased in diabetic rats, but the total capacity for net zinc absorption was
not affected (161). In human patients with type II diabetes mellitus, zinc-
tolerance curves were less (i.e., the increase of serum zinc after an oral zinc
dose was less), suggesting that zinc absorption may be impaired in this
disease (225). Dexamethasone increased zinc uptake but not intestinal me-
tallothionein uptake in adrenalectomized rats (28). Copper absorption was
enhanced in rats bearing Dunning mammary tumors and was inversely pro-
portional to the amount bound to a lO,OOO-dalton component, presumably
254 ROBERT J. COIJSINS VOl?(?)IP 65

metallothionein (72). Estrogen had the reverse effect on these parameters.

Mason et al. (262) showed that substantial amounts of copper in the intestines
of neonatal rats were bound to a metallothionein-like protein. These levels
were decreased substantially by day 15 postpartum.

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).

III. PLASMA TRANSPORT

A. Copper

Plasma copper is distributed among three major constituents comprising

two pools between which it does not appear to be exchangeable (257, 414).
Ceruloplasmin represents a tightly bound pool that accounts for at least 90%
of the total plasma copper in most species (175, 188). However, in some
species, e.g., the chicken, plasma ceruloplasmin is particularly low (411).
Albumin- and amino acid-bound copper constitute the less tightly bound
pool, which may increase in copper content directly after a meal. Albumin
does not bind copper in the dog, notable because this species is particularly
susceptible to copper toxicity (103). Albumin and/or amino acids are believed
to be the ligands that transport copper in the portal circulation (155, 258,
458). This strongly suggests that loosely bound copper is the form donated
to hepatocytes. The transport mechanism may require an equilibrium among
cell receptors, albumin, and free copper. Albumin may have different binding
sites for copper and zinc (291,326). Proton displacement data on the interaction
of copper and zinc with the imidazole side chains of the 16 histidine residues
of albumin suggest that the two strongest copper-binding sites are not iden-
tical to the two strongest zinc-binding sites (345). Morgan (289) has described
a histidine-rich glycoprotein from serum that has a higher binding affinity
for copper than for zinc. Its molecular weight is 58,000, a size that suggests
it could be mistaken for albumin with protein separation methods based on
size alone.
Copper secreted from hepatocytes (parenchymal cells) is principally in
the form of ceruloplasmin. Extrahepatic copper is probably presented to
tissues in this form. Owen (311) and Campbell et al. (52) demonstrated that
copper uptake into tissues was closely related to release of ceruloplasmin
from the liver. Marceau and Aspin (257) found that copper derived from
albumin was readily removed from the plasma, whereas copper from ceru-
loplasmin was not. Presumably, albumin-bound copper was readily ex-
changeable with tissue copper. Hsieh and Frieden (200) showed that ceru-
loplasmin restored cvtochrome c oxidase activity in copper-deficient, rat,s to
a greater extent than intravenous administration of copper complexes of
albumin, histidine, or CuC12. Similarly, Harris and DiSilvest,ro (184) showed
that the activity of lvsvl oxidase in chick aort,a was directly correlated with
ceruloplasmin contentof the plasma. Ceruloplasmin was the major copper
protein found in human amniotic fluid (158).
Copper bound to low-molecular-weight moieties may also he donated to
tissues. This could be most important when the dietary copper supply is
diminished or in copper-relat,ed genetic diseases, e.g., Wilsons disease (55).
Both conditions involve a reduction in circulating ceruloplasmin. Bioflavonoids
have been suggested as a chelated form of plasma copper that could donate
copper to tissues (102). Pickart and Thaler (328) have reported that glycyl-
histidyllysine stimulated copper uptake bv cultured hepatoma cells and con-
comitantly stimulated DNA synthesis and cell proliferation. Glycylhisti-
dyllysine was found associated with albumin and cu-globulin fractions of
human plasma.
Illness and trauma can markedly influence the plasma copper level. This
change is usually upward; however; during dietary copper deficiency, total
parenteral nutrition, Menkes disease, and frequently in Wilsons disease,
decreases in serum copper are reported. Owen (31.4i extensively reviewed
these changes in serum copper. Hypocupremia has been identifiedin patients
receiving pharmacological doses ;,f zinc orally (334). The literature is filled
with examples of increases in plasma copper in response t,o infection, in-
flammation, trauma, etc. (for reviews see refs. 19,332). Elevated c.eruloplasmin
levels usually elevate plasma copper. Ceruloplasmin is classified as an acute-
phase protein; thus during these altered phvsiological st.at,es, its secretion
into the plasma would be expect.ed. Some of these inflammation-related in-
creases may be in response to common chronic diseases, e.g., rheumatoid
arthritis (402) and periodontal disease (153, 424). The mechanism that prob-
ably accounts for this is complex and involves stimulation of ceruloplasmin
synthesis in and/or secretion from liver cells in response to physiological
stimuli. The hormonal basis for the regulation of ceruloplasmin synthesis
and secretion is discussed in section IVG.

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.

IV. GENERAL F~JNCTIONS OF COYPER AND ZINC:

Nutrition experiments have clearly . shown that dietary copper is required

for a variety of functions, including bone formation, proper cardiac function,
260 ROBERT J. COUSINS Volt4 3n e 65

connective tissue development, myelination of the spinal cord, keratinization,

and tissue pigmentation (48,314,433). Dietary fructose may have an influence
on copper status during marginal copper intakes and therefore affect these
functions (152). In most cases the function of copper in metalloenzymes
involves electron transfer and enzymatic binding of molecular oxygen: The
hepatic copper export protein ceruloplasmin has been extensively studied.
The enzymatic functions of this metalloprotein are discussed below (sect.
VIIA). Superoxide dismutase is another extremely important and well-studied
copper metalloenzyme (268). The cytoplasmic form of this enzyme contains
zinc and copper. It is abundant in the liver as well as in other tissues. It
catalyzes the dismutation of the superoxide anion 0, + 0, + 2H+ -+ HZ02 +
Oz. Valentine and Pantoliano (436) have suggested the possibility of a role
for superoxide dismutase related to intracellular copper and/or zinc metab-
olism. Another copper-dependent enzyme, lysyl oxidase, is important in the
biosynthesis of connective tissue. Its deficit probably accounts for the lesions
of copper deficiency that affect bone and connective tissues (297). Its activity
in the liver was related to collagen synthesis during hepatic fibrosis (382).
Some knowledge of the regulation of synthesis of these copper metalloenzymes
is emerging. However, understanding of exactly how and when copper is
inserted into the intact apoenzyme molecule is still fragmentary.
There are more zinc than copper metalloenzymes in liver. Because these
enzymes have been derived and studied from numerous species, it is difficult
to establish from the literature the exact number of zinc-containing hepatic
enzymes in a given species. However, of the more than 100 enzymes that
require zinc for maximum catalytic activity, a substantial number are in
liver. In this role, zinc provides structural integrity to the enzyme and/or
participates directly in catalysis. Examples of zinc metalloenzymes from
liver include DNA and RNA nucleotidyl transferases; alcohol dehydrogenase;
glutamic, lactic, and malic dehydrogenase; and Saminolevulinic acid dehy-
dratase (104). Some of these enzymes are influenced by dietary zinc status,
whereas others are refractory to alterations in the dietary zinc supply (24,
227, 228). Guzelian et al. (176) have demonstrated a 95% loss of 6-aminole-
vulinic acid dehydratase activity in rat hepatocytes maintained in primary
culture with zinc-depleted media. Repletion with zinc restored activity to
100% of the initial value. These changes correlated directly with the cellular
zinc content. These experiments show the exciting potential of the isolated
hepatocyte culture system for studies on the relationship between cellular
metal status and hepatic metalloenzyme activity.
The multiplicity of functions in which zinc is involved is clearly due to
the role this metal plays in specific metalloenzyme systems. Zinc has important
roles in bone formation, cell-mediated immunity, generalized host defense,
and a wide variety of factors related to tissue growth. An interesting corollary
to the direct involvement of zinc in metalloenzymes is its potential role as
an allosteric regulator of enzyme activity. Pedrosa and co-workers (322) have
suggested, based on in vitro experiments, that fructose 1,6-bisphosphatase
A pri1 19X.5 COWER AND ZINC METAI.WLISM 261

is regulated by zinc. Zinc is a strong inhibitor of the enzyme. An intracellular

zinc chelat,or such as histidine could remove the metal from the allosteric
regulatory site and thus increase activity. Fructose 1,6+isphosphatase is a
constitutive enzyme that is sensitive to various physiological stimuli (471),
many of which are also regulators of hepatic zinc metabolism (98). These
event,s could be related, but only correlations are currently possible. It is
also possible that zinc influences this enzyme in a classic zinc: metalloenzymic
manner (22).
Chvapil (70) and Rettger and ODell (24) have suggest.ed an alternative
role of zinc in cellular function, i.e., a paramount role in membrane structure.
The latter envision that this role could be equally important to zinc metal-
loenzymes in understanding the plethora of signs in zinc-deficient animals.
As w&h copper, little is known about the mechanism byc which zinc is in-
corporated into macromoIecules, i.e., enzymes or membranes.

V. HEPATIC METABOLISM OF COPPER AND ZINC

Copper concentrat,ion in the liver is influenced by the dietary copper

supply as well as by specific disease factors. Basic aspects of copper metabolism
by hepatocytes are shown in Figure 4. Generally, when the dietary copper
level is low, the liver copper concentration is diminished and vice versa (as
reviewed in ref. 433). Parenteral and oral doses of copper also markedly
influenced the amount of copper accumulated by the liver (33, 34, 36, 37, 41,
429,457). Hepatic copper was significantly increased in streptozotocin-induced
diabetic rats (133). In humans, liver coI;per levels were found to increase in
Wilsons disease and lead to tissue damage (413) and primary biliary cirrhosis
(204). Sheep are particularly sensitive to copper and can accumilate toxic
quantities of this nutrient ih the liver (38). Copper toxicity in dogs may be
related to the failure of albumin to bind copper and thus influence hepatic
uptake (360).
TJnfortunately little is known about the copper uptake mechanism in
hepatocytes. AS ientioned earlier, amino acids and/or albumin is probably
the ligand from which copper is presented to hepatocytes for transport across
the plasma membrane. It is likely that an equilibrium is established between
amino acid-bound and albumin-bound copper, which influences how much
copper is accumulated by hepatocytes. In vitro evidence that such an equi-
librium exists has been developed (188). Although it is difficult to separate
the relative importance of amino acids versus albumin in hepatic copper
uptake, it is interesting that Marceau and Aspin (258) demonstrated that
intracellular radioactivity from [4Cu]albumin was bound to a soluble liver
 Fhcrptor
I\\
/. /
I
/
Glucocorticold - - - - - - - -, - - / /cm
\ -, - IAAA
/ /
\\
II /
t /\ I t \ \ \ I/ ; ,/

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

protein of 10,000 daltons. In comparison, 67Cu from [67Cu]ceruloplasmin was

bound to 30,000-dalton species. It has been shown that the hepatic uptake
of copper from the blood is rapid and efficient. Initial liver uptake was >50%
in rats and >80% in humans (307, 311). Owen (314) has reviewed this area
in detail.
Saltman et al. (360), in the first detailed analyses of the hepatic copper
accumulation process in vitro, found that uptake followed first-order kinetics.
The saturability of the uptake mechanism in these experiments with rat
liver slices was interpreted to imply that specific sites on or in the cell were
responsible for the accumulation-binding phenomenon. Copper binding by
the slices was affected by temperature but was not influenced by either
sulfhydryl inhibitors or dinitrophenol. No effect on 64Cu accumulation was
noted when the liver slices were incubated with any of the following: zinc,
nickel, magnesium, cobalt, and iron. It was hypothesized that one or more
copper chelates were responsible for donating the metal to the cellular binding
sites. Using a similar rat liver slice technique, Harris and Sass-Kortsak (183)
demonstrated that copper uptake was facilitated by amino acids. This process
was abolished under anaerobic conditions, but it did not appear to require
metabolic energy production, because 2,4-dinitrophenol had no effect on copper
uptake.
The influence of the endocrine system on copper metabolism is well
documented (147) and is briefly reviewed in section VII.
Weiner and Cousins (457) have studied the accumulation of copper in
primary monolayer cultures of rat liver parenchymal cells. This culture pro-
cedure allows the chronic action of hormones to be developed and precludes
carryover effects related to preexisting hormonal or nutritional status of the
rats from which the cells were obtained (231). Copper accumulation from
serum-free medium was temperature dependent and strongly inhibited by
cyanide and N-ethylmaleimide but not by 2,4-dinitrophenol or NaN3. Initial
uptake was rapid, then a slower linear accumulation phase was observed for
12 h. That was followed by steady uptake efflux of copper, suggesting equil-
ibration with exchanging pools. Incubation of the hepatocytes with cyclo-
heximide substantially increased copper accumulation from the medium,
whereas incubation with actinomycin D had no effect. Efflux experiments
demonstrated that copper was readily sequestered by intracellular compo-
nents and was not available for rapid export from the cell. Incubation under
conditions of O-20 PM zinc had no influence on copper accumulation. This
agreed with the findings of Saltman et al. (360). Similarly, copper (O-20 PM)
had no influence on zinc accumulation by these cells.
Schmitt et al. (367) reported that copper uptake by freshly isolated rat
hepatocytes in suspension exhibited saturation kinetics with a K, of 10.6
PM and a Vmaxof 2.8 nmol Cu min. l mg- protein. Similar kinetic parameters
were obtained whether the copper was presented as a free ion or as a 1:l
copper-histidine complex. It was proposed that copper is transported as the
free ion. The finding that copper uptake was substantial at 20C and nearly
the same at 0C suggests that a non-temperature-dependent binding effect
may account for some of the uptake observed. In contrast to findings by
others (360, 459), this group found that copper and zinc were mutually an-
tagonistic with respect to uptake (367). Albumin markedly inhibited copper
uptake by a substrate-removal mechanism. It was suggested that incubation
of hepatocytes with albumin may limit the free copper available to the cells.
However, hepatocvtes must exchange nutrients with plasma albumin in situ;
thus albumin mai be an unavoidable competitor for plasma copper. Collec-
tively these data suggest that copper uptake is a specific process that follows
a passive carrier-mediated mechanism that may utilize free or albumin-
bound copper. Hepatocvte suspensions prepared from hemizvgous and het-
erozygous brindled mide accumulated less copper than normal hepatocytes
(90). A defect in a copper-specific excretory . process was suggested as the
responsible mechanism.
Copper accumulation bv primary cultures of rat liver parenchvmal cells
is stimulated in response to hormones (458). Epinephrine had themost pro-
nounced effect in this respect. When the cells were incubated with phen-
oxybenzamine (an a-adrenergic blocker) the epinephrine response was abol-
ished. Glucagon had less effect on copper accumulation in these cells. The
addition of dexamethasone to these cell cultures stimulated zinc accumulation
but had only a small influence on copper accumulation (457). Estradiol-17fl
and testosterone actually appeared to lower copper accumulation. The hor-
monal regulation of copper metabolism has inputs that are markedly similar
to mechanisms that influence the transport of amino acids (221) and the
intracellular redistribution of calcium (288). With copper the normal plasma
concentration is between 15 and 20 PM, whereas the free amino acid con-
centrations are usually in the millimolar range. Enhanced membrane trans-
port of the amino acids that chelate copper mav result in increased uptake
of the metal as well. Nevertheless, because hormones rarely have unilateral
actions, it would not be unique for a hormone to exert independent effects
on the transport of a trace element such as copper, separate from distinct
effects on the transport of amino acids or other plasma constituents.
The demonstration that copper accumulation bv liver cells was responsive
to hormones suggests that changes in hepatic uptake could occur during
altered physiological states. In support of this, streptozotocin-induced diabetic
rats exhibiting hypoinsulinemia and hyperglucagonenlia were found to ac-
cumulate a substantial amount of copper and zinc in both liver and kidney
(133). In contrast, although endotoxemia has been shown to increase both
insulin and glucagon levels (218), liver zinc was found to increase in hamsters
after endotoxin treatment, whereas liver copper was unchanged (117). In
view of the generally deleterious efiects that excess copper has on tissues,
the possible effect that changes in hormones, e.g., epinephrine and glucagon,
have on copper transport deserves further study. It is important in evaluating
this work to attempt to separate effects on copper due to actual membrane
April 1985 COPPER AND ZINC METABOLISM 265

transport from those related to intracellular changes that influence the

amount of copper accumulated by liver cells.

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

--+n Cu- Metallothionein

Zn- Metallothionein
Plasma -
Metallothloneln

Amino Acid%
April 1985 COPPER AND ZINC METABOLISM 267

cellular zinc content. Within 20 h in culture at 16 PM, the accumulation of

extracellular 65Zn represented 67% of the total cellular zinc content. These
data suggest that complete zinc turnover in liver parenchymal cells occurs
in 30 h at normal plasma zinc concentrations. At least 90% of the total cell
zinc was accounted for by a slow phase of uptake that may represent an
exchange mechanism. The exchange process was saturable with a K, of 9.5
PM and Knax of 10 pmol Zn. min- mg- protein.
l The fast phase of 65Zn
uptake primarily represented net accumulation. Comparisons of the kinetics
for the fast pool suggests it may be a precursor to the exchange process.
Glucocorticoids appeared to increase the slow exchange rate with a concom-
itant increase in the fast-pool size.
A most relevant finding with the cultured liver cell model was the dem-
onstration by Failla and Cousins (131) that zinc accumulation exchange could
be stimulated in response to glucocorticoids. Previous work by Cox (83) with
HeLa cells clearly showed that hormones with glucocorticoid activity would
stimulate zinc uptake. The findings with liver cells, however, provided the
first demonstration that this uptake stimulus may have physiological rele-
vance at the hepatic level. Zinc uptake in response to the glucocorticoid
dexamethasone was over twofold after 20 h in culture. In this system other
hormones, including sex steroids and some polypeptide hormones, had no
influence on overall zinc accumulation (131). Accumulation in most of these
studies was longer than a few hours and therefore was the result of processes
that innuence uptake and cellular efflux. The coordinated effects of glucagon
and glucocorticoids on specific hepatic processes, e.g., gluconeogenesis via
enhanced amino acid transport and phosphoenolpyruvate carboxykinase syn-
thesis, are well characterized (221, 231). Glucagon was shown to stimulate
zinc uptake and/or exchange by liver parenchymal cells (237). The effect is

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

additive, when combined with that of dexamethasone, in a way that is similar

to stimulation of the gluconeogenic pathway. The glucagon effect was not
blocked by insulin. The latter may even enhance its effects. These hormonal
actions on zinc metabolism suggest that changes in liver zinc metabolism
are collectively mediated, at least partly, by glucagon, insulin, and gluco-
corticoids. A depression in serum zinc and hepatic zinc accumulation are
well-documented acute effects of stress, infection, and trauma and could be
mediated by these hormones (see sect. v1B4).
Studies with humans with either 65Zn or 6gmZn have shown that orally
administered zinc was rapidly absorbed, with the major portion initially
being taken up the liver (1, 151, 404). Kinetic analysis indicates rapid- and
slow-exchange phases between plasma and liver (151). These findings agree
with data from experiments with isolated liver parenchymal cells (130). A
kinetic model for rats yielded comparable data. Liver had the highest transfer
coefficient of all tissues (204).
The influence of hormones on zinc metabolism in humans has been known
for decades. Note that long-term corticosteroid therapy in patients decreased
serum zinc concentrations (148). Recently, Henkin and associates (189) es-
tablished that hepatic zinc metabolism was altered in humans given glu-
cocorticoids in a way that was predictable, based on data from the rat he-
patocyte model (131). The early observations in this area have been well
reviewed (291).

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

to metallothionein (33, 258, 443). Cytoplasmic zinc is similarly subject to

metabolic changes. Of the total zinc taken up by livers of calves orally dosed
with 65Zn, 55% was in the soluble fraction; this increased to 75% when 600
ppm zinc was fed (222). Dramatic changes in hepatic cytosol zinc content of
rats were found after administration of zinc parenteral by or after zinc
repletion (350). Bremner and co-workers (35, 38) and Chen et al. (61) made
similar findings.
Weser and Bischoff (460) observed that intraperitoneal injection of 65Zn
resulted in greater uptake of 65Zn by rat liver nuclei relative to the other
subcellular fractions. This suggests that early in the zinc uptake process, a
mechanism may exist to shunt a substantial amount of newly acquired zinc
to the nucleus, for specific functions within that organelle. Although the data
are from nuclei of calf thymus rather than liver, Bryan et al. (46) found that
0.1 pg Zn/mg DNA and 0.025 Fg Cu/mg DNA were tightly bound to chromatin.
Both metals were present in higher-molecular-weight oligonucleosomes. Ev-
idence was obtained that copper was associated with a subset of nucleoprotein
complexes and may participate in higher levels of chromatin organization.
Most nuclear zinc was also associated with both high- and low-molecular-
weight precipitable nucleoprotein. These findings are compatible with roles
for copper in higher-level chromatin organization and with the role of zinc
in specific aspects of cell division and RNA metabolism. Convincing evidence
has been presented with the EugZenagracilis model that zinc has widespread
roles in chromatin structure and metabolism, including that of histones (266,
438). These findings probably relate directly to the function of zinc in gene
expression in mammalian cells.
It is generally assumed that intracellular zinc and copper are partitioned
in two general ways, i.e., as components of metalloenzyme systems and, at
least in the case of zinc, with macromolecules, particularly membranes. These
metalloenzymes have been the subject of detailed reviews and, for the sake
of brevity, are discussed only cursorily.
Understanding of the metabolism of hepatic copper and zinc stems largely
from in vivo and in vitro experiments with radioisotopes. Enteral or parenteral
administration of 64Cu results in a rapid hepatic accumulation of this ra-
dionuclide (307, 311, 314). Gel-filtration chromatography of the cytosol
(100,000-g supernatant) results in resolution of 64Cu into two major chro-
matographic peaks. These correspond to molecular weights of 35,000 and
10,000. The larger molecular-weight fraction may contain superoxide dis-
mutase and other copper metalloenzymes. The smaller molecular-weight peak
was accounted for primarily as metallothionein (33). Van den Hamer (443)
reported with intact rats that after administration, copper was probably
first associated with a lower-molecular-weight protein (metallothionein), and
with time, copper appeared to be transferred to higher-molecular-weight
species. With increasing doses of copper, more copper was recovered in a
particulate fraction. The nature of this fraction was not clear, but it may
well be highly polymerized copper-rich metallothionein (33). Newly accu-
mulated 64Cu was bound principally to metallothionein in rat liver paren-
270 ROBERT J. COUSINS Vohme 65

chymal cells in primary monolayer culture (457). These uptake experiments

could be interpreted as a demonstration of the critical function of metal-
lothionein in the initial uptake and/or retention of cellular copper. Alter-
natively they could be a reflection of binding to available copper sites.
The kinetics of hepatic 65Zn metabolism have been well studied in animals
and humans (1,204,222,284). Richards and Cousins (347,348) demonstrated
that intraperitoneal administration of zinc resulted in a marked increase in
hepatic zinc uptake. Because the increase in hepatic zinc occurred simulta-
neously with the incorporation of labeled amino acids into metallothionein,
it was proposed that zinc uptake into the liver under these conditions involved
increased metallothionein synthesis. Notable in these studies was the ob-
servation that zinc uptake was prevented when rats had received prior ad-
ministration of actinomycin D, a known inhibitor of mRNA synthesis. In
these early experiments it was postulated that metallothionein induction
involved increased transcription of the gene that codes for the protein (348,
367). Early experiments by Winge et al. (466) suggested that acute admin-
istration of copper increased expression of the gene for a copper-binding
protein. Bremner et al. (41) and Riordan and co-workers (255, 354) have
provided convincing evidence that this copper protein is metallothionein.
Sciortino et al. (372) established that metallothionein was present in both
parenchymal and nonparenchymal (Kupffer) cells of rat liver.
The localization of metallothionein within tissues has relied primarily
on immunochemical methods. Kojima and Hamashima (233) performed the
first localization study with rabbit antisera. Using an immunofluorescent
technique, they found that kidneys from a variety of species contained me-
tallothionein. Banerjee et al. (13) found detectable amounts of metallothionein
in the cytoplasm of hepatocytes in subsequent similar studies. Cadmium
treatment was shown to increase the amount of metallothionein in the sub-
cellular structures. A salient observation was the appearance of immuno-
reactive material in the nuclei of cells from cadmium-treated animals (13,
63). Danielson et al. (89) carried out a similar localization study and reported
that the protein appeared both within cells and at extracellular sites. The
extracellular localization was particularly prevalent in the liver sinusoids
in animals that had been treated with large amounts of cadmium. The surface
columnar epithelial cells of intestinal villi also reacted positively to the me-
tallothionein antibody. Based on the extracellular localization of the protein
after cadmium treatment, this group suggested metallothionein may have a
transport or excretory function.

C. Copper and Zinc Eflux From Hepatocytes

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

storage, substantial disulfide formation may occur.) 4) No aromatic amino

acids occur in the peptide. 5) The protein exhibits a significant degree of
polymorphism. 6) There is excellent sequence homology, indicating a highly
conserved primary structure. There are two major isometallothioneins, des-
ignated metallothioneins I and II. These differ slightly in amino acid com-
position and are separable by DEAE ion-exchange chromatography or gel-
permeation high-performance liquid chromatography (HPLC) (340,422). The
DNA sequence data indicate there may be more than two metaliothionein
genes (214), which could explain the numerous isometallothioneins found in
tissue extracts via reverse-phase HPLC (230). The physical properties of
metallothionein have been discussed in detail in the review edited by Kagi
and Nordberg (209).
Configuration of the unique metal-binding sites of metallothionein has
attracted considerable attention. Initial studies based on primary structure
suggested that cysteinyl residues provided the necessary metal-binding li-
gands (232). More recent experiments have suggested that each metal ion is
arranged in a tetrahedral coordination complex with four thiolate ligands
(308). These 13Cd-NMR (nuclear magnetic resonance) studies suggest that
the seven metals (cadmium or zinc) bound to metallothionein are arranged
in two clusters, one with four atoms (cluster A) and another with three
atoms (cluster B). Metal binding appears to occur first in cluster A, followed
cooperatively by cluster B. The order of metal release may be the reverse of
binding (294). When copper is bound, the coordination arrangement is prob-
ably different and may involve more atoms per mole (21). This could account
for the markedly different metabolic properties of copper-rich metallothi-
oneins discussed in section VI&D.
Metallothioneins have been isolated from a variety of species. The most
extensive characterizations have been developed from liver, kidney, and in-
testine. Although a great deal of attention has been given to its relationship
to cadmium toxicology, when it was isolated from adult liver, metallothionein
was shown to be predominantly a zinc-containing protein (47,350). The fact
that it binds zinc and copper under physiological conditions suggests that
the protein is involved in the metabolism of both nutrient metals. Human
fetal liver metallothionein contained primarily zinc when isolated from the
soluble fraction, but more copper was associated with metallothionein when
recovered from the particulate fraction (354, 358). Similarly, large amounts
of liver metallothionein were found in livers of rats and sheep during the
pre- and neonatal period (20, 40, 331). Earlier, Morel1 et al. (287) and Porter
and Hills (331) isolated copper-rich proteins that were probably metallo-
thioneins.

B. Synthesis

The biosynthesis of metallothionein is controlled by complex processes.

A list of physiological factors and experimental conditions that induce liver
metallothionein is shown in Table 2.
274 ROBERT J. COUSINS Volume 65

I. Regulation by metals

Piscator (329) first developed the concept that metallothionein is an

inducible protein. He demonstrated that cadmium administration to rabbits
resulted in a substantial increase in the hepatic metallothionein content. He
further proposed that the teleological basis for this induction was to protect
the animal from cellular damage resulting from administration of this toxic
heavy metal. Shaikh (376) demonstrated that cadmium administration to
rats could increase the incorporation of labeled amino acids into hepatic
metallothionein, suggesting that de novo protein synthesis was involved in
this induction. Webb (454) made similar observations with cadmium and zinc
treatments and proposed a translational mechanism for regulation. Subse-
quent to these early observations, Squibb and Cousins (405,407) and Richards
and Cousins (347) demonstrated that cadmium and zinc, respectively, were
able to stimulate the incorporation of labeled amino acids into metallothi-
onein. These double-labeled experiments showed that biosynthesis of the
protein and accumulation of the metal bound to metallothionein were linked

TABLE 2. Physiological factors and experimental conditions

that result in metallothionein induction

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

on a close temporal basis. Significant in these studies was the demonstration

that actinomycin D completely blocked the ability of these metals to induce
synthesis and bring about accumulation of the metal. It was proposed that
these inductive events involved differential gene expression that altered the
metallothionein mRNA population in the tissues studied. Subsequent ex-
periments, developed partly because of the rapid increase in understanding
of the mechanism of gene expression, have substantiated this hypothesis.
Our experiments and those of Bremner, Brady, Cherian, Klaassen, Shaikh,
Whanger, and others (reviewed in ref. 209) have provided interlocking evidence
that metallothionein is induced by copper, cadmium, and zinc. Winge and
co-workers (466) initially reported the characterization of a distinct, non-
metallothionein copper-rich protein from livers of copper-injected rats. This
putative species termed copper chelatin, which is now generally considered
to be artifactual, appeared to have a lower cysteine content and different
elution behavior on DEAE ion-exchange chromatography compared to me-
tallothionein. Subsequent studies by Bremner and co-workers (41) clearly
demonstrated that copper-rich metallothioneins could be isolated from rat
and pig liver when anaerobic conditions were used to preclude alteration
(particularly oxidative) of native metallothionein. Numerous other studies
have verified the influence of copper on metallothionein production by the
liver (36, 209). In support of this work it was demonstrated that antisera to
the copper-induced protein would precipitate either the copper-induced protein
or cadmium-induced metallothionein, suggesting essentially identical anti-
genie sites for both proteins (255). The discrepancy between these observations
and those that led to the chelatin theory could relate to oxidation of me-
tallothionein sulfhydryls; atypical amino acid analysis due to oxidative
changes catalyzed by Cu2; or, as that suggested in a reevaluation of the
initial chelatin hypothesis, a contaminant isolated with copper metallothi-
onein that influenced the amino acid analysis (465). In vitro evidence showed
that reduction of Cu2+ to Cu+ in the presence of glutathione led to a copper-
zinc replacement in zinc metallothionein (422). A copper-zinc replacement
was shown in vivo as well (95). Moreover, based on hybridization to a me-
tallothionein cDNA probe (log), copper treatments increased the amount of
metallothionein mRNA. These data collectively provide strong support for
the contention that copper induces metallothionein as do zinc, cadmium,
glucocorticoids, glucagon, and epinephrine. It is also conceivable that copper
or other metals replace zinc from existing metallothionein, and then the
newly released zinc activates the metallothionein gene promoter. Copper
metallothioneins have been isolated from yeast (50, 149), and gene fusion
experiments show that activation of these genes has a specific requirement
for copper (50). These findings may be related to the unresolved copper me-
tallothionein-chelatin issue in mammalian cells.
Squibb et al. (407) demonstrated that metallothionein synthesis, as judged
by pulse labeling with [3S]cystine, reached the maximum rate -5-7 h after
parenteral administration of zinc (containing 65Zn). Subsequent to this in-
276 ROBERT J. COUSINS Volume 65

crease in biosynthetic rate there was increased incorporation of 65Zn from

the administered dose into the newly synthesized protein. Both actinomycin
D and cordycepin blocked the incorporation of 35S-labeled amino acids into
metallothionein. These data further established that the induction of me-
tallothionein by zinc involved increased transcription of the metallothionein
gene. Subsequent work involving measurement of metallothionein mRNA
has resulted in induction kinetics similar to those obtained in these pulse-
labeling studies. Chen et al. (62) proposed a similar estimate of the rate of
metallothionein synthesis after cadmium administration.
A major step in understanding the relationship of metallothionein syn-
thesis to gene expression was the use of cell-free translation systems to
measure metallothionein mRNA levels in nucleic acid extracts of various
tissues. The wheat germ translation system was shown to translate metal-
lothionein mRNA with considerable fidelity (8, 378). Poly(A)+ RNA used to
program the translation system was isolated from rat liver polyribosomes.
Metallothionein mRNA levels after administration of zinc were found to
closely parallel the rates of metallothionein synthesis (379, 407). Methods
by which the cell-free products were characterized varied widely. Shapiro
et al. (379) used covalent chromatography with activated thiol Sepharose
chromatography, whereas Andersen and Weser (8) employed SDS-PAGE.
Antibodies directed against purified metallothioneins have been used to im-
munoprecipitate metallothionein produced in these systems (64, 248). More
recently, cell-free translation systems have provided evidence that metal-
lothionein mRNA levels in intact animals are increased during fetal and
neonatal development (7), copper administration (278), hormone treatments
(116), endotoxin administration (100, 117), IL-l (loo), isopropanol (427), and
dietary increases in zinc (269, 276).
Induction kinetics for metallothionein mRNA in kidney in response to
zinc and cadmium was similar to that reported in liver (109, 425). Shapiro
and Cousins (378) analyzed the polyribosomal distribution of metallothionein
mRNA in rat liver and found that virtually all of this mRNA pool was
associated with free polyribosomes. The localization of metallothionein mRNA
with free polyribosomes strongly suggests that it is not a secreted protein
and that its biological role is related to an intracellular process. Cherian et
al. (64) confirmed the free polyribosomal origin of metallothionein mRNA.
Andersen and Weser (8) estimated the size of mRNA at 9s via sucrose gradient
fractionation. Subsequent experiments involving glyoxalation of mRNA en-
riched in metallothionein coding regions and separation by electrophoresis
through 2% agarose gels indicated that metallothionein mRNA was between
280 and 390 nucleotides (377). This estimate of size for the final processed
transcript is compatible with 181 nucleotides for the protein coding sequence
and a poly(A) tail of 100-200 nucleotides. This indicates that the coding
region is sufficient for only a single metallothionein polypeptide and, fur-
thermore, that the mRNA is not polycistronic.
The unique inducibility of metallothionein by heavy metals suggested
that this protein was an excellent candidate for studies involving regulation
April 1985 COPPER AND ZINC METABOLISM 277

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

Previous sections have emphasized the large volume of data suggesting

that administration of significant amounts of copper or zinc via parenteral
or enteral routes will lead to the induction of metallothionein in intestines,
liver, kidney, and other tissues. An equally large volume of literature exists
on the induction of this protein in these organs in response to acute and
chronic exposure to the toxic metal cadmium (209). However, the latter area
is beyond the scope of this review.
The most exciting development in the area of the regulation of metal-
lothionein biosynthesis relates to the induction of this protein as a response
to physiological stimuli. When the dietary supply of zinc or copper is elevated
sufficiently, induction of metallothionein occurs, primarily in the liver and
intestine, and is followed by increased accumulation and/or redistribution
of these metals in both tissues. This physiological response has been examined
278 ROBERT J. COUSINS Vohme 65

in two ways: short-term fluctuations in dietary concentration and longer-

term feeding of high levels over an extended period. A rationale can be
developed for each approach, although the human diet is more likely to vary
markedly in trace element content from day to day compared with that of
well-maintained domestic animals.
Acute changes in the dietary zinc concentration can alter hepatic me-
tallothionein-bound zinc. Experiments with rats demonstrated that hepatic
and intestinal metallothionein levels were normally low (0.4 and 1.4 pg
Zn/g, respectively). However, a l-day zinc-deficient diet (<I mg Zn/kg) de-
creased metallothionein to only trace levels (350). Repletion with a semi-
purified diet containing 150 mg Zn/kg increased metallothionein-bound zinc
in both organs (10.7 and 12.5 pg Zn/g, respectively). A further l-day depletion
reduced these levels again. Similarly, when rats were fed the zinc-deficient
diet for 7 days and subsequently repleted with 25, 75, or 150 mg Zn/kg diet
for 1 day, both hepatic and liver metallothionein increased in relation to the
dietary level. Results of these experiments could be interpreted to indicate
three things: I) metallothionein is rapidly synthesized to bind potentially
deleterious actions of extra Zn2+ (detoxification), 2) zinc binding to metal-
lothionein is part of an intracellular function in which the binding protein
serves as an intermediate or coordinating ligand to accommodate an influx
of zinc into the intracellular pool, or 3) a combination of both 1 and 2.
Whanger and co-workers (61,302) have shown that relatively large amounts
of zinc must be fed over extended periods (weeks) before substantial amounts
of hepatic metallothionein accumulate. In one 8-wk feeding study, appreciable
amounts of metallothionein-bound zinc did not appear in the soluble fraction
of rat liver until at least 1,000 mg Zn/kg diet was fed. These high metal-
lothionein-zinc levels were reduced to basal levels within 3 days when the
diet was changed to one deficient in zinc. Supplementation with 2,000 mg
Z&kg diet has been shown to markedly increase the labeling of the protein
with [14C]cystine (302).
The temporal relationship between metallothionein mRNA levels and
metallothionein induction with realistic dietary levels of zinc has been studied.
In these experiments rats were depleted of their dietary zinc supply for a
4-day period and then fed by gavage a diet containing 125 mg Zn/kg (269).
In response to this dietary treatment, the serum zinc concentration increased
dramatically for up to 3 h and then returned toward normal levels, which
were not reached until 18 h after the feeding. As the serum zinc concentration
declined, the liver cytosol zinc content steadily increased. A portion of this
was zinc associated with metallothionein. A considerable amount of the zinc
taken up by the liver was probably derived from the diet, because inclusion
of 65Zn with the test meal resulted in substantial amounts of radioactivity
in the cytosol fraction of the liver. Pulse labeling with [35S]cystine of rats
fed 65Zn-containing diet demonstrated that the dietary induction of metal-
lothionein in liver appeared to involve expression of genes for both metal-
lothionein I and II. Changes in polyribosomal metallothionein mRNA levels
April 1985 COPPER AND ZINC METABOLISM 279

slightly preceded changes in the augmented rate of metallothionein synthesis

in liver. Similar findings were made for the induction of intestinal metal-
lothionein, but increased metallothionein mRNA levels in intestine preceded
those changes found in the liver by a few hours (276). This would be expected
because the intestine is the first tissue to encounter an augmented dietary
zinc supply.
Food restriction in rats has been shown to increase liver metallothionein-
bound zinc (35, 349). There was a lo-fold increase in these levels in rats fed
either zinc-adequate or zinc-deficient diets immediately prior to the fast
(349). In zinc-depleted rats the fast increased the serum zinc concentration,
suggesting mobilization of zinc stores, perhaps from muscle. Actinomycin D
administration blocked the induction of liver metallothionein in response to
food restriction, suggesting that increased metallothionein gene expression
accounted for the induction. The primary inducer could be either Zn2+ or one
or more hormones associated with the physiological response to a reduction
in food intake. These findings are important because any experimental treat-
ment that drastically decreases food consumption might be expected to con-
comitantly induce liver metallothionein. This response to fasting can be
broadly viewed as an indication of the sensitivity of this system to mild
metabolic changes. Perhaps metallothionein induction is part of a defense
mechanism in which cellular zinc is conserved during a reduction in the
dietary supply, or it may represent the need to augment a specialized function
for the protein. Moreover, plasma metallothionein levels may be reflective
of body zinc status.

3. Hormonal regulation

Several papers published around 1978 further placed the physiological

regulation of metallothionein on a sound experimental basis. Oh and co-
workers (303) demonstrated the induction of metallothionein in liver and
kidney after exposure to a variety of stresses, including cold, heat, burn,
strenuous exercise, and Ccl, intoxication. Sas and Bremner (364) made similar
observations with CC& inhalation in chicks fed a zinc-supplemented diet.
Chicks fed a zinc-deficient diet did not respond. Ohtake and co-workers (305)
correlated liver regeneration after partial hepatectomy with metallothionein
synthesis. They also showed that induction of DNA synthesis in liver by
isoprenaline and glucagon injections was correlated to metallothionein syn-
thesis, and they speculated that the latter was important to supply zinc for
enzymes required for DNA synthesis. Sobocinski et al. (394) correlated the
induction of hepatic metallothionein with the hypozincemia induced in rats
by SalmoneLLa infection. They suggested that the apparent flux of plasma
zinc to the liver was related to hepatic zinc redistribution. These findings
were correlated with the concomitant block by actinomycin D of hepatic zinc
accumulation and metallothionein induction in animals intraperitoneally in-
jected with zinc (348, 349).
280 ROBERT J. COUSINS Volume 65

Metallothionein induction by stress and fasting was related to hormonal

control at the cellular level when Failla and Cousins (130,131) demonstrated
with isolated rat liver parenchymal cells in primary culture that glucocor-
ticoids would enhance zinc uptake and induce metallothionein. The fact that
most of the extra zinc accumulated was as metallothionein, an effect blocked
by actinomycin D, provided strong evidence that the glucocorticoid dexa-
methasone could regulate the metallothionein gene expression in hepatocytes.
Zinc accumulation and metallothionein synthesis were demonstrated to be
dose dependent. Dexamethasone increased the slow zinc exchange rate with
a concomitant increase in the labile zinc pool. This suggests that metallo-
thionein induced by glucocorticoids is a component of the labile pool of zinc
in hepatocytes (319). Metallothionein induction in response to this gluco-
corticoid was shown in HeLa cells (213). Earlier experiments by Cox (83)
demonstrated the effect of glucocorticoids on protein synthesis-dependent
zinc uptake in HeLa cells. These data support the hypothesis that metal-
lothionein induction in response to physiological stimuli partly involves al-
tered regulation of metallothionein gene expression in response to gluco-
corticoid hormones. After administration of dexamethasone to intact rats,
enhanced levels of hepatic metallothionein mRNA were observed, followed
by an increased rate of metallothionein synthesis and concomitant depression
in the serum zinc concentration (116).
The effect of glucocorticoids on metallothionein synthesis in isolated
cells and intact rats afforded a model to examine steroid hormonal regulation
of metallothionein gene expression. Hager and Palmiter (177) demonstrated
that dexamethasone administration to mice would increase metallothionein
mRNA levels and rate of metallothionein mRNA synthesis (transcription).
These mRNA levels were 1,350 molecules per cell 6 h after hormone treatment.
Basal levels usually averaged 13 molecules per cell but occasionally increased
to a level of 300 because of stress associated with husbandry conditions. A
cDNA probe to rat metallothionein mRNA was used in similar studies with
rats and confirmed the effect of glucocorticoids on metallothionein mRNA
levels (6). Similar cDNA hybridization techniques demonstrated that zinc
and dexamethasone act additively to increase the hepatic levels of metal-
lothionein mRNA (342). Moreover, similar estimates of metallothionein
mRNA levels were obtained by translational assay or [35S]cystine pulse la-
beling of metallothionein. The apparent additive response between dexa-
methasone and zinc agrees with gene amplification experiments. These showed
that glucocorticoid regulation was lost while regulation by metals was retained
(265). This result suggests that control segments for the gene are at different
positions in the 5 direction from the coding region.
Another series of experiments led to the suggestion that the hormonal
induction of metallothionein is not solely under glucocorticoid control (115).
Current knowledge of both steroid and peptide hormone action on numerous
processes, e.g., carbohydrate metabolism (231), makes this probable. Specif-
ically, glucagon administration was shown to increase hepatic metallothionein
April 1985 COPPER AND ZINC METABOLISM 281

(115). When dexamethasone was administered simultaneously with glucagon,

there was a short-lived transient synergistic effect of these two hormones
with respect to the amount of zinc associated with hepatic metallothionein.
Similarly, glucagon was shown to have an independent effect on zinc accu-
mulation and metallothionein synthesis by rat liver parenchymal cells in
primary culture. Glucagon and dexamethasone together significantly aug-
mented the response observed with either hormone alone (237). These changes
occurred within the same general temporal sequence as increases in glucose
production and phosphoenolpyruvate carboxykinase activity occur in response
to the hormones (231). Glucagon administration to intact rats had no effect
on serum zinc concentrations. However, the induction by glucagon of hepatic
metallothionein was blocked by cycloheximide but not by actinomycin D (99,
100, 115). Furthermore glucagon stimulated [35S]cystine incorporation into
metallothionein. Dexamethasone given with glucagon produced an additive
increase of r5S]cystine incorporation into metallothionein. Hepatic mRNA
derived from polysomes showed increased metallothionein mRNA levels in
glucagon-treated rats, especially when given with dexamethasone. Metal-
lothionein mRNA levels in the total RNA extract did not show increases of
the same magnitude. Therefore glucagon may act through translational reg-
ulation to promote translation of preexisting metallothionein mRNA.
Experiments with isolated hepatocytes suggest that both glucagon and
epinephrine have additive effects with glucocorticoids on zinc uptake and
metallothionein. However, in these experiments the effects of epinephrine
and glucagon are blocked by actinomycin D, suggesting transcriptional control
(79). This has been made more tenable with the identification of a CAMP-
sensitive sequence of the promoter of the phosphoenolpyruvate carboxykinase
(195). A similar coding sequence may impart CAMP sensitivity to the me-
tallothionein genes. Brady and Helvig (32) have provided data suggesting
that both epinephrine and norepinephrine administration increase metal-
lothionein-bound zinc. Only the epinephrine treatment was additive with
that of dexamethasone. Collectively the evidence that glucagon and epi-
nephrine have an independent effect as weil as an additive effect with glu-
cocorticoids suggests that they have an extremely important role in altering
zinc metabolism as they do in the regulation of glucose metabolism.

4. Stress factors and mediators

Acute infection and endotoxemia have been shown to have marked effects
on copper and zinc metabolism (reviewed in refs. 17, 332). Some of these
changes could be due to decreased food consumption, a condition that, as
noted above, leads to hepatic metallothionein induction. Acute endotoxin
administration increased 65Zn uptake into the livers of chicks and rats, par-
ticularly as zinc bound to metallothionein in the soluble fraction (117, 223,
364, 392). Subsequently, Etzel et al. (117) examined the effect of E. coZi and
Bacteroides meZaninogenicus endotoxins on zinc and copper metabolism in
282 ROBERT J. COUSINS Volume 65

hamsters and confirmed the relationship between hepatic metallothionein

concentration and hypozincemia. Metallothionein mRNA levels were elevated
to four times the levels found in controls 6 h after E. coZi endotoxin was
given. B. meZaninogenicus endotoxin had only a small effect on serum zinc
and liver metallothionein. Concurrent increases in serum copper and copper
deposition in gingival tissues were greater with this endotoxin. The effect
of E. coli endotoxin on serum zinc depression, liver metallothionein mRNA,
and metallothionein-bound zinc was similar in fasted rats and zinc-depleted
rats (100). Sobocinski and Canterbury (392) have suggested that the response
of hepatic zinc metabolism to endotoxin and IL-l occurs through a common
mediator, which they propose is glucagon. However, exogenous glucagon
administration does not lead to a reduced serum zinc concentratioh, although
it does have a marked effect on metallothionein synthesis (100, 115).
Inflammation induced by turpentine administration has been shown to
induce hypozincemia and hepatic metallothionein synthesis. Adrenalectomy
did not influence these parameters (392, 393). Isopropanol administration
had similar effects in normal and adrenalectomized rats (427). Moreover this
inflammatory-like response did not affect copper metabolism but induced
hepatic metallothionein mRNA levels comparable to those observed with
high doses of zinc. Isopropanol significantly increased plasma glucagon levels,
with a peak 4 h after treatment. However, because the isopropanol effect
was independent of the adrenal gland and glucagon did not produce hypo-
zincemia (a characterization of acute inflammatory response), other media-
tors, specifically IL-l, are probably also involved. Recently, DiSilvestro and
Cousins (100) demonstrated with rats that IL-l administration would sig-
nificantly increase liver metallothionein mRNA levels, accompanied by an
increase in metallothionein and hypozincemia. Cycloheximide totally blocked
these IL-l effects; however, they were not totally inhibited with actinomycin
D. In contrast, serum zinc levels were not diminished in adrenalectomized
rats after an IL-l injection, but there was a modest increase in liver me-
tallothionein zinc. Interleukin 1 induced hypozincemia and substantial
amounts of metallothionein in adrenalectomized rats when given simulta-
neously with dexamethasone. The induction of this hepatic protein in response
to IL-l is interesting, because this agent may play a role in increasing the
metallothionein level in certain tissues as part of the host-defense mechanism.
Interleukin 1 may act partly to influence hormonal status (16, 17, 332).
Recent evidence suggests that an alternate mechanism is possible to partially
explain its effect on metallothionein gene expression in intact animals. Baracos
et al. (14) have reported that IL-l leads to muscle protein breakdown through
a PGEB-induced activation of lysosomes. This occurred simultaneously with
fever, which augmented the catabolic process. Interleukin 1 and endotoxin
also enhance the flux of amino acids from muscle to liver (16, 19, 332, 452).
Large amounts of zinc are present in muscle, and urinary zinc output may
serve as an index of muscle catabolism (140). Therefore it is intriguing to
speculate that as muscle is broken down in response to IL-l, sufficient amounts
April 1985 COPPER AND ZINC METABOLISM 283

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

or whether induction via a hormonal stimulus leads to increased metal ac-

cumulation. (For further studies see refs. 354, 358, 467.)

C. Degradation

The degradation of metallothionein is as important in the overall turnover

of this protein as the rate of synthesis; however, the latter has received far
more attention (see Fig. 5). Nevertheless most estimates suggest that me-
tallothionein is a fairly rapidly turned over protein. Zinc repletion-depletion
experiments with rats demonstrated that 24 h was sufficient to decrease a
substantial pool of metallothionein-bound zinc to negligible levels (350). Chen
et al. (61) made similar observations in rats fed very high levels of zinc. Oh
et al. (302) followed the degradation of [35S]cystine-labeled metallothionein
induced by feeding 2,000 mg Zn/kg and calculated a T1i2 of 1.3 days, whereas
without this dietary treatment the T 1/2 was 3.4 days. Feldman and Cousins
(137) observed that the Tl12 of zinc-induced liver metallothionein was between
18 and 20 h. Both zinc and the protein were labeled to allow simultaneous
estimation of metal and protein turnover. In those experiments, the 35S-
labeled hepatic proteins from the soluble fraction had a collective Z1,2 of 4
days. Both isometallothioneins I and II appeared to degrade at the same
rate. With a similar approach to investigate the degradation of liver cadmium-
induced metallothionein, the cadmium form of the protein, which contained
cadmium and zinc (Z:l), was shown to have a ?!I/2 of ~3.5 days (139). Within
the 9 days that the experiment was conducted, there appeared to be virtually
no turnover of cadmium in the protein. Cadmium can remain associated with
the protein for months (353,461). Because the metallothionein was obviously
degraded, as indicated by the turnover of the labeled polypeptide, it was
concluded that Cd2+ was released during degradation but that Cd2+ has a
sufficiently high binding constant to allow it to rebind immediately to nascent
thionein (apometallothionein) polypeptide chains (78, 139).
Bremner et al. (37) reported that the Yii2 of 35S-labeled copper-induced
metallothionein was 12-17 h, depending on dietary zinc status. Similarly,
Prins and Van den Hamer (337) found 35S-labeled metallothionein was readily
degraded in brindled mice. The method to measure degradation in these
studies involved chromatography of the soluble fraction of liver; this may
account for the apparent fast in vivo T 1/2 discussed below. Since this initial
work, a wide variety of physiological and nutritional factors have been shown
to alter the Z1,2 in intact-animal experiments. Whanger and Ridlington (461)
have reviewed the data on metallothionein degradation in many species.
Feldman et al. (138) investigated the degradation of metallothionein
under in vitro conditions. For this work 35S-labeled zinc metallothionein and
cadmium metallothionein were prepared in vivo from zinc- and cadmium-
injected rats, respectively. Also, metal-free metallothionein (thionein) was
prepared and its degradation examined. Under the in vitro conditions em-
ployed, both trypsin and Pronase substantially degraded the protein, but to
April 1985 COPPER AND ZINC METABOLISM 285

different degrees. When these metallothioneins were subjected to hydrolysis

under the influence of lysosomal proteases (at pH 5), it was found that zinc
metallothionein was less resistant to degradation than was cadmium me-
tallothionein. Note that thionein was degraded extremely rapidly by either
neutral or lysosomal proteases. Studies by Held and Hoekstra (187) suggest
that the copper metallothionein produced by adding exogenous copper to
purified metallothionein was resistant to degradation by lysosomal proteases.
Bremner and Mehra (39) also could not show appreciable degradation by
lysosomal proteases of copper metallothionein produced in vivo.
The combination of in vivo and in vitro degradation data provides a
possible explanation for altered accumulation patterns associated with hepatic
metallothioneins. The marked susceptibility of thionein to degradation by
nonlysosomal proteases suggests its degradation could be cytosolic (216) and/
or lysosomal. Similarly, limited proteolytic activity of nonlysosomal proteases
for zinc and cadmium metallothioneins suggests that some degradation of
these could be cytosolic. If a function of metallothionein requires degradative
release of bound metal, a slow cytoplasmic process would be expected. A
cytoprotective role would also dictate this. The data suggesting that Cd
from degraded cadmium-containing metallothionein is rebound to nascent
metallothionein polypeptides support such a protective role.
The susceptibility of zinc metallothionein and cadmium metallothionein
to lysosomal protease activity, in the same order as apparent degradation
in vivo, suggests the lysosome could be the major site of metallothionein
degradation. It may be relevant that zinc has been shown to inhibit proteolysis
in liver lysosomes (272). The degradative behavior of copper metallothionein
is at variance with this concept. In vivo evidence suggests it has an apparent
Z1,2 that is shorter than the zinc-containing protein (37). In contrast, copper
metallothionein is resistant to in vitro degradation by lysosomal proteases,
perhaps because it more easily undergoes polymerization. Bremner and Mehra
(39) have reviewed the evidence bearing on this inconsistency. Numerous
possible explanations exist. One relates to the methods used to initially isolate
metallothionein in the in vivo experiments. A number of studies have shown
that metallothionein-like proteins accumulate in lysosomes under conditions
associated with high hepatic copper levels. For example, Johnson et al. (207)
fractionated livers of Bedlington terriers (with inherited copper toxicosis)
with low-speed centrifugation and isolated a copper-rich (7-8 atoms/molecule)
metallothionein from a particulate fraction. Therefore it is likely in the in
vivo degradation studies that copper metallothionein was removed from the
soluble fraction during centrifugation prior to its quantitation by gel-filtration
chromatography. This would obviate an accurate measurement of T1/2, and
it suggests that the estimates of copper-metallothionein degradation made
with this approach are erroneous. However, copper metallothionein associated
with the particulate fraction of liver from copper-injected rats had a Y& of
25 h, compared to the T 1/2 in the cytosol (21 h). This suggests that lysosomal
degradation of copper metallothionein occurs in vivo (39). Nevertheless the
286 ROBERT J. COUSINS Vdume 65

in vitro evidence well explains the in vivo degradative behavior of cadmium

and zinc metallothioneins. Therefore the bulk of the evidence favors a major
role for lysosomal proteases in metallothionein degradation. This role could
be to handle excess metallothionein and may not be related to normal turnover
mechanisms associated with the intracellular role(s) of the protein.
The minimal turnover of copper metallothionein could explain why, under
certain conditions, substantial amounts of copper are accumulated in tissues.
The situation is similar to that observed with the accumulation of cadmium.
However, copper is a far more ubiquitous atom in biological systems. Diseases
that lead to the accumulation of abnormal intracellular amounts of copper,
e.g., Wilsons disease (415) and experimental diabetes (133), could result
partly from accumulation of copper as poorly degraded copper metallothionein
formed, in abundant amounts, in response to altered cellular uptake or efflux
processes.

D. Function

The exact function(s) of metallothionein in intestine, kidney, liver, and

perhaps other tissues remains to be defined. Metallothionein gene expression
is regulated by the metals that the protein binds, suggesting a function for
it in trace-element metabolism. The binding phenomenon could be for the
singular purpose of maintaining homeostasis of zinc and copper (319). Other
functions are possible, however. A role as a donor of zinc and copper ions
to apometalloproteins has been proposed based on in vitro data. Numerous
apoenzymes have been tested for reactivation on addition of metallothionein.
Zinc metallothionein reactivated apo forms of carbonic anhydrase (242,432),
alkaline phosphatase and aldolase (432), and copper metallothionein reac-
tivated apo forms of tyrosinase and hemocyanin (21). Reactivation of a
mammalian apometalloenzyme by copper metallothionein has not been dem-
onstrated. Because the mechanism by which metals are incorporated into
metalloproteins has not been elucidated, the hypothesis that metallothionein
has a role in this process is tenable. During a period of stress, increases in
the activity of many metalloenzymes may be called for, and this role for the
protein fits within the framework of a host-defense process. Donation of
bound metals to cell membranes is also a possible function within this context.
A correlation between metallothionein induction and host defense has
emerged in experiments with adjuvant-induced arthritis in rats. Simkin (383)
has presented clinical evidence for a positive therapeutic effect of oral zinc
supplements in treatment of human rheumatoid arthritis. Similarly, zinc
treatment diminished the inflammatory signs associated with adjuvant ar-
thritis in rats (426). 130cis-Retinoic acid (13-cis-RA) was also found to reduce
inflammation in this model (45). When zinc and RA treatments were combined,
there was an additive effect on inflammation reduction coupled with an
increase in serum ceruloplasmin and liver metallothionein (426). These ob-
April 1985 COPPER AND ZINC METABOLISM 287

servations suggest that zinc could have an ameliorative influence on inflam-

mations such as those associated with inflammatory disease. Thus metal-
lothionein induction could be a reflection of the role zinc and/or the protein
plays in cellular defense mechanisms. The extent of induction could be a
reflection of the severity of the disease and/or the effectiveness of therapeutic
strategies. A relevant finding was that metallothionein synthesis in mac-
rophages responds to inducing metals or glucocorticoids and that this has
been correlated to a decrease in toxicity of the cells to endotoxin (318). Since
zinc may affect phagocytic activity (71), metallothionein could provide a
regulatory function in this regard.
A cytoprotective role analogous to metal resistance factors could be
envisioned in which its sole function is related to metal binding and detox-
ification. Zinc can inhibit the adenylate cyclase activity of hepatocyte mem-
branes (254), which would have obvious consequences if not regulated or
prevented.
The sulfhydryl-rich structure of metallothionein has led to experiments
designed to investigate its potential as a binding moiety and perhaps detoxifier
of hepatotoxins (51). Intraperitoneal administration of a variety of alkylating
agents induced hepatic metallothionein in rats (234). Some of the induction
was the result of decreased food intake caused by the toxins. The induction
of metallothionein by iodoacetate was blocked by cycloheximide, suggesting
increased metallothionein synthesis. Because these experiments were in intact
animals, indirect induction via hormonal stimuli cannot be ruled out. Sim-
ilarly, zinc given intraperitoneally reduces liver damage and mortality in
endotoxin-treated rats (395). Endresen and co-workers (112) have suggested
that the resistance to anticancer drugs in some lines of cultured mammalian
cells was related to binding of these alkylating agents to metallothionein.
The drug resistance that was demonstrated could be related to higher-than-
normal levels of metallothionein-gene expression. It is also possible, however,
that alkylation of existing metallothionein in situ frees bound zinc, which
could reactivate the gene promoter and thus increase synthesis and possibly
augment the detoxification cycle. Similarly, in other experiments, detoxifi-
cation of alkylating compounds and resistance to certain compounds in cul-
tured cells is correlated with concomitant high cellular metallothionein levels
(l&374,430). This further suggests that in some forms of drug therapy, this
defense mechanism may be counterproductive. Consumption of large amounts
of dietary copper and/or zinc over extended periods may influence these
mechanisms by changing tissue metallothionein levels. Hepatic metallothi-
onein binds gold (380), and therapies related to gold salts (as anti-inflam-
matory agents) may be limited or augmented by high metallothionein levels.
Platinum from antineoplastic drugs containing this metal may also bind to
metallothionein, which could influence the effectiveness of platinum-con-
taining drugs (12, 472). This important area of metallothionein-related de-
toxification needs further exploration in view of its potential role in drug
detoxification and/or resistance.
288 ROBERT J. COUSINS Vdume 65

VII. CERULOPLASMIN

A. Properties and Functions

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

ceruloplasmin, suggesting a beneficial effect of elevated ceruloplasmin levels.

Also notable is a demonstration of ceruloplasmin synthesis and secretion by
Sertoli cells of the testes (384).
The function of ceruloplasmin as an antioxidant is an attractive hy-
pothesis, because the generation of oxidation products, including 0; and
H202, is associated with conditions that increase plasma ceruloplasmin levels.
For example, the oxidation of ferrous iron leads to superoxide ion. This in
turn can lead to peroxidative damage. Ceruloplasmin, through its ferroxidase
activity, can catalyze the oxidation of Fe2+ with concomitant production of
H20 from 02, lowering the likelihood of superoxide generation by Fe2+. The
antioxidant activity of plasma has been related to ceruloplasmin (105). Gold-
stein et al. (167) have shown that ceruloplasmin can serve as a scavenger of
superoxide radicals. The protein may have direct anti-inflammatory properties
(96). It can also inhibit metal ion-induced lipid peroxidation (105). The protein
also has a protective effect on copper-stimulated erythrocyte lysis, a function
that is not related to superoxide scavenging (252). Frieden (155) has estimated
that the collective radical-scavenging potential of ceruloplasmin in serum is
less than that in superoxide dismutase. Because ceruloplasmin is extracellular
and superoxide dismutase is primarily intracellular, ceruloplasmin could be
a major scavenger in plasma, particularly when its concentration is elevated.
The integration of this function of ceruloplasmin, with its classification as
an acute-phase reactant, is discussed below.

B. Synthesis and Secretion

The hormonal factors that have been shown to influence ceruloplasmin

production by the liver are shown in Figure 4. The ceruloplasmin gene has
not been as extensively studied as many other genes that are expressed in
liver cells. Gaitskhoki et al. (156, 157) reported that ceruloplasmin mRNA
activity was associated only with membrane-bound polyribosomes. Each
mRNA molecule was translated simultaneously by 16-18 ribosomes. This is
at the low end of the number of ribosomes that would correspond to the
80,000-dalton size proposed for the intracellular ceruloplasmin precursor.
Ceruloplasmin-synthesizing polyribosomes were identified with anticerulo-
plasmin immunoglobulins and were used to prepare ceruloplasmin-enriched
mRNA. The latter was used as a substrate for reverse transcription to syn-
thesize a cDNA for hybridization studies. Hybridizations revealed that ce-
ruloplasmin mRNA accounted for 0.29% of the total poly(A) RNA. Gaitskhoki
et al. estimated the abundance of ceruloplasmin mRNA at 400-535 molecules
per liver cell, which suggests it is not a low-abundance message. Most of
these molecules (90%) were polyribosomal, with the remaining 10% being
located in the postribosomal supernatant. In vitro translation of liver poly(A)
RNA with the wheat germ system yielded an 84,000-dalton product on im-
munoprecipitation and SDS-PAGE. Addition of rat liver membranes to the
translation reaction mixture converted this product into 80,000- and 65,000-
290 ROBERT J. COUSINS Volume 65

dalton fragments. These were designated as proceruloplasmins, whereas the

initial 84,000-dalton cell-free product was designated as preproceruloplasmin
(339). A 65,000-dalton precursor accumulated in the Golgi. It was proposed
that ceruloplasmin maturation involves ligation of two precursor fragments.
Although this seems unlikely, Prozorovski et al. (338) have emphasized that
the sequence homology in each half of holoceruloplasmin supports this sug-
gestion. However, Takahashi and co-workers (428) have shown that there is
a triplicate of sequence homology. As discussed below, the levels of plasma
ceruloplasmin fluctuate, indicating that ceruloplasmin gene expression may
be regulated by physiological and pharmacological stimuli, as are genes for
other acute-phase proteins.
Ceruloplasmin synthesis and/or secretion is altered by inflammation,
hormones, and copper. A list of the physiological factors and experimental
conditions that increase plasma ceruloplasmin levels is shown in Table 3. It
is generally recognized that plasma concentrations of acute-phase globulins,
including ceruloplasmin, increase after tissue injury, localized acute inflam-
mation, and chronic inflammatory diseases such as rheumatoid arthritis.
Reports of this stress-related effect have appeared over the last 50 years.
The past literature has been reviewed recently (332). The data suggest ce-
ruloplasmin serves in a host-defense role, as Denko (96) proposed. Its prop-

TABLE 3. Physiological factors and experimental conditions

that increase plasma ceruloplasmin levels

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

* Also called leukocytic endogenous mediator and endogenous pyrogen.

April 1985 COPPER AND ZINC METABOLISM 291

erties as a radical scavenger and copper-donor are particularly attractive in

this respect. Most evidence for increased synthesis during stress stems from
ceruloplasmin-related increases in serum oxidase activity. However, exper-
iments that used immunoprecipitation methods to actually quantitate ce-
ruloplasmin molecules suggested that these changes are due to altered rates
of synthesis (57, 197, 458). Roeser et al. (355) found increased serum copper
and ceruloplasmin ferroxidase activity in rheumatoid arthritis patients. Sim-
ilar increases were observed in patients with periodontal inflammation (gin-
givitis) (153, 424). Meyer et al. (281) reported that that turpentine-induced
serum ceruloplasmin and copper levels were independent of the adrenal
glands. Copper-deficient rats are more susceptible to acute inflammatory
agents (X2), but the copper needed for increased synthesis of ceruloplasmin
does not deplete liver copper stores (73). Milanino and Velo (283) have reviewed
in detail the relationship between serum ceruloplasmin and acute inflam-
mation.
Denko (96) has provided evidence supporting a relationship between a
reduction in the inflammatory response in rats and ceruloplasmin admin-
istered by injection. He presented this as evidence supporting the anti-in-
flammatory action of ceruloplasmin in rheumatic diseases. Recently the effect
of intraperitoneal administration of zinc in reducing paw edema associated
with adjuvant-induced arthritis was demonstrated (426). This experimental
inflammation increased ceruloplasmin levels, which were further increased
after zinc treatment. Previously, Brinckerhoff et al. (45) had found that high
doses of 13-cis-RA had a similar effect in the same model for arthritis.
Combined administration of both zinc and RA reduced paw edema to a greater
extent than either treatment alone, while producing a greater increase in
serum ceruloplasmin (426). These experimental data from an animal model
support the clinical findings of Simkin (383) that pharmacological doses of
zinc may have a beneficial effect on rheumatoid arthritis. Furthermore the
data support the hypothesis that ceruloplasmin synthesis, secretion, or turn-
over is altered in response to various agents as a defense against inflammation
and its metabolic consequences (96). The effect of RA is undoubtedly phar-
macological and probably unrelated to vitamin A status, because vitamin A
deficiency does not affect intracellular ceruloplasmin (346). Nevertheless,
vitamin A deficiency could influence the ceruloplasmin level in plasma.
Infection is considered a factor that increases ceruloplasmin synthesis.
Starcher and Hill (411) showed that SalmonelLa infection in chickens markedly
increased serum ceruloplasmin. Curtis and Butler (87) have documented the
increase in serum ceruloplasmin produced in chickens by E. coli endotoxin
administration. Cycloheximide partially blocked this increase. The immediate
increase in ceruloplasmin was attributed to a release of the intact protein
from liver cells. Curtis and Butler reasoned that subsequent increases were
related to hormonal induction of ceruloplasmin synthesis. Etzel et al. (117)
found that E. coli endotoxin injections into hamsters produced a biphasic
response on serum copper content and ceruloplasmin, as measured by SDS-
292 ROBERT J. COUSINS Vdume 65

PAGE. This suggests an initial immediate release of hepatic ceruloplasmin

followed by de novo synthesis. Moreover they found that endotoxin treatment
was accompanied by pronounced increases in copper content of the gingival
copper levels. Note that B. melaninogenicus endotoxin, isolated from dogs
with gingivitis, produced greater increases in both serum copper and gingival
copper and less of an effect on other parameters than did E. coli endotoxin.
This suggests that endotoxins from different sources may influence cerulo-
plasmin levels to different degrees.
Meyer et al. (281) presented early experimental data showing hormonal
regulation of ceruloplasmin synthesis and secretion in experiments designed
to explain serum ceruloplasmin increases in stress conditions. They found
that both epinephrine and estradiol increased ceruloplasmin levels in rats
via a mechanism that did not require the adrenal gland. Earlier, estrogen
was shown to increase the serum copper content in humans (356). Both ACTH
and hydrocortisone increased serum ceruloplasmin in chickens (411). Based
on this observation, Starcher and Hill (411) proposed that any stress-related
change in ceruloplasmin involves adrenal steroids. An mRNA-related mech-
anism was proposed to explain the differential in production. Curtis and
Butler (87) found that both ACTH and P-methasone (a glucocorticoid) would
increase serum ceruloplasmin in chickens. They attributed the effect of en-
dotoxin on serum ceruloplasmin to ACTH and glucocorticoid action. Epi-
nephrine or norepinephrine injected into chicks had similar inductive ef-
fects (154).
A single injection of ACTH increased serum ceruloplasmin in rabbits
(5). In contrast, Evans and Wiederanders (127) found that ACTH given as
multiple injections depressed plasma ceruloplasmin and copper levels. Sim-
ilarly, Lahey et al. (238) reported that ACTH injections depressed serum
copper levels in leukemia patients. Early experiments by Gubler et al. (174)
showed that ACTH did not lower plasma copper levels of rats in which the
plasma copper was elevated by turpentine treatment. Ceruloplasmin levels
are elevated in the developing human neonate (468), which could be related
to changes in growth hormone levels, because hypophysectomy elevated he-
patic copper (60, 192). Preterm infants were found to exhibit a precocious
increase in serum ceruloplasmin (327). Chang et al. (57) could not detect
serum ceruloplasmin in newborn pigs. They observed increases in cerulo-
plasmin, independent of dietary copper content, during the 1st wk of life
and attributed this to initiation of apoceruloplasmin synthesis. Growth hor-
mone could have signaled this induction, but it was not tested.
Thyroid hormone administration or hyperthyroidism increased serum
copper levels in humans (281, 292). To the contrary, thyroxine injections
decreased both plasma copper and ceruloplasmin in rats (60, 119), but hy-
pothyroidism in humans was accompanied by decreased serum copper (192).
Early studies by Lahey et al. (238) documented a correlation between preg-
nancy and elevated serum ceruloplasmin. This finding has been extended to
include women taking oral contraceptives (356) and could be linked to copper
April 1985 COPPER AND ZINC METABOLISM 293

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

concentrations of copper used in these experiments produced hemolysis and

a general increase in hepatic synthesis of plasma proteins. Moreover they
observed with copper-deficient rats that orally administered copper increased
the rate of ceruloplasmin synthesis ([3H]leucine as a pulse) without affecting
the synthesis of plasma or liver proteins in general. Copper-adequate rats
did not increase ceruloplasmin synthesis in response to copper. It was sug-
gested that the increase in synthesis observed by Evans et al. (124) may
have been related to copper-induced hemolysis.
Because all experiments on the regulation of ceruloplasmin levels and
on the apparent synthesis of ceruloplasmin were conducted with intact an-
imals, Weiner and Cousins (457-459) utilized the rat liver parenchymal cell
system to examine these parameters in an isolated system free of the plethora
of endogenous stimuli. These studies showed that 4Cu accumulated by mono-
layer cultures of cells was continuously subject to efflux. A major portion of
the copper released was ceruloplasmin. Actinomycin D and cycloheximide
increased the apparent accumulation of copper. Cordycepin had a similar
effect, further demonstrating that RNA processing to mRNA was required
to transfer copper out of hepatocytes. It was suggested that these increases
in cellular copper content occurred because steps in ceruloplasmin synthesis
and/or processing are inhibited, thus precluding the vehicle needed for copper
release. Previously, Gregoriadis and Sourkes (172) showed an increase in
hepatic copper with inhibitors of various phases of protein synthesis. However,
these inhibitors also could have affected secretion of biliary components, a
condition that would alter the interpretation of these findings.
To measure changes in ceruloplasmin synthesis by liver parenchymal
cells directly, rabbit antisera to highly purified rat ceruloplasmin was prepared
(458). Cells were incubated with 3H-labeled amino acids that collectively were
~46% of the amino acid residues of rat ceruloplasmin (256). [3H]ceruloplasmin
was immunoprecipitated from the culture medium and cell lysates. The SDS-
PAGE indicated that the secreted protein was the 130,000-dalton protein
expected for intact ceruloplasmin. Dexamethasone significantly increased
intracellular and secreted [3H]ceruloplasmin by 1.3- and 2.0-fold, respectively.
However, the glucocorticoid did not significantly affect cellular copper ac-
cumulation. Epinephrine had the most pronounced effect on 64Cu uptake from
the medium (see sect. VA), doubling the 64Cu incorporation into intracellular
ceruloplasmin. This increase was paralleled by increased 3H-labeled amino
acid incorporation into ceruloplasmin. Conversely, epinephrine did not have
a major effect on either 64Cu or [3H]ceruloplasmin secretion from the liver
cells. The [3H]ceruloplasmin content of both cell cytosol and the culture media
was increased when the extracellular copper content was raised to physio-
logically relevant concentrations. This is consistent with the hypothesis of
Holtzman and Gaumnitz (197) that the ceruloplasmin decay rate is related
to copper presence in the protein. Incubation with 50 PM for 12 h or more
significantly increased [3H]ceruloplasmin secretion by the cells, which sug-
April 1985 COPPER AND ZINC METABOLISM 295

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

alter translational regulation. Physiologically, regulation of synthesis and

secretion by epinephrine and glucocorticoid is attractive and could partly
explain the hormonal stimuli involved in increasing the plasma levels of this
protein.

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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