1998 - Copper and Zinc in Inflammatory and Degenerative Diseases

Copper and Zinc in Inflammatory and
Degenerative Diseases
Copper and Zinc in
Inflammatory and
Degenerative Diseases
Edited by
K.O. Rainsford
Division of Biomedical Sciences, School of Science and Mathematics,
Sheffield Hallam University, Sheffield, UK
R. Milanino
Institute of Pharmacology, University of Verona, Verona, ltaly
J.R.J. Sorenson
Department of Pharmacy, University of Arkansas, little Rock, AR, USA
and
G.P. Vei o
Institute of Pharmacology, University of Verona, Verona, Italy
.....
"
SPRINGER SCIENCE+BUSINESS MEDIA, BV.
Library of Congress Cataloging-in-Publication Data is available.
ISBN 978-94-010-5757-8 ISBN 978-94-011-3963-2 (eBook)

DOI 10.1007/978-94-011-3963-2
Printed an acid-free paper
AII Rights Reserved

© 1998 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 1998
Softcover reprint of the hardcover Ist edition 1998
No part of this publication may be reproduced or utilized in any form or by any means, electronic,
mechanical, including photocopying, recording or by any information storage and retrieval system,
without written permission from the copyright owner.
Contents
List of Contributors vii

Preface ix
1 History of zinc therapy
TU Hoogenraad
2 Physiological properties of copper and zinc
V Albergoni 7
3 Biological chemistry of copper compounds
L-O Klotz and U Weser 19
4 The biological chemistry of zinc
S Rahuel-Clermont and MF Dunn 47
5 Copper and zinc metallothioneins
V Albergoni and E Piccinni 61
6 Zinc in the regulation and therapy of inflammatory diseases and
gastrointestinal ulceration
KD Rainsford and B Zeitlin 79
7 Copper complexes for therapy of cancer and autoimmune diseases
JRJ Sorenson ll3
8 Zinc and copper in the treatment of rheumatic diseases
F Fernandez-Madrid 125
9 Topically applied copper preparations for anti-inflammatory therapy
SJ Beveridge l39
10 Regulation by copper of rat adjuvant-arthritis: a model of chronic
inflammation especially suitable for studying the mechanisms
of copper anti-inflammatory activity
R Milanino, M Marrella, GP Velo, P Cristofori and A Terron 147
11 Copper and zinc compounds and cell surface interactions
ME Davies and M Pasqualicchio 161
12 Copper and postmenopausal osteoporosis
JJ Strain 173
l3 Menkes disease: a genetic defect of copper transport
B Sarkar 179
Index 189
v
List of Contributors
VALBERGONI L-OKLOTZ
Department of Biology Physiological Chemical Institute
University of Padova Eberhard-Karls-Universitiit
Via Trieste 75 Hoppe-Seyler Strasse 4
1-35121 Padova D-72076 Tiibingen
Italy Germany
SJBEVERIDGE RMILANINO
Department of Chemistry Institute of Pharmacology
Central Coast Campus University of Verona
University of Newcastle Policlinico di Borgo Roma
Brush Road 1-37134 Verona
Ourimbah, NSW 2258 Italy
Australia
EPICCINNI
ME DAVIES Department of Biology
Strangeways Research Laboratory University of Padova
Worts' Causeway via U. Bassi 58/B
Cambridge, CB14RN 1-35121 Padova
UK Italy
MFDUNN S RAHUEL-CLERMONT
Department of Biochemistry Department of Biochemistry
University of California at Riverside University of California at Riverside
Riverside, CA 92521-0121 Riverside, CA 92521-0121
USA USA
F FERNANDEZ-MADRID KD RAINSFORD
Division of Rheumatology Division of Biomedical Sciences
Hutzel Hospital School of Science and Mathematics
4707 St Antoine Boulevard Sheffield Hallam University
Detroit, MI 48201 Pond Street, Sheffield, SI lWB
USA UK
TU HOOGENRAAD BSARKAR
Department of Neurology Department of Biochemistry Research
University Hospital Utrecht The Hospital for Sick Children
Heidelberglaan tOO 555 University Avenue
3508 GA Utrecht Toronto, Ontario, M5G lX8
The Netherlands Canada
vii
COPPER AND ZINC IN INFLAMMATORY AND DEGENERATIVE DISEASES
JRJ SORENSON UWESER

Department of Pharmacy Physiological Chemical Institute
University of Arkansas for Medical Eberhard-Karls-Universitat Tiibingen
Science Campus Hoppe-Seyler Strasse 4
4301 W. Markham, Slot 522 D-72076 Tiibingen
Little Rock, AR 72205 Germany
USA
BZEITLIN
JJSTRAIN Division of Biomedical Sciences
Human Nutrition Research Group School of Science and Mathematics
University of Ulster Sheffield Hallam University
Coleraine, BT52 I SA Pond Street, Sheffield, SI IWB
Northern Ireland UK
viii
Preface
It has now been well established that changes occur, albeit variably, in the
plasma and tissue concentrations of both copper and zinc in a large number of
acute and chronic inflammatory and degenerative diseases. These metal ions
form associations with an immense array of enzymes and regulatory macro-
molecules such that it is not hard to envisage that there will be consequences for
their activity as a result of alterations in the metabolism and distribution of
copper and zinc. Yet this simplistic view belies more complex issues. Thus,
variations in the cellular and circulating levels occur with different conditions
and at different stages in their progression. The well-known opposing changes of
copper and zinc as well as their contrasting actions makes for many of the
puzzling aspects in the understanding of the role of these metals in inflamma-
tory andn degenerative diseases.
We have tried here to bring together the current state of knowledge of the
roles of copper and zinc in the pathophysiology of various chronic diseases,
their importance in various cellular and biochemical processes, and the
application of various complexes of zinc and copper in treating a range of
diseases. The mode of action of these metal complexes is considered in detail.
This book should be of interest to a wide readership ranging from basic
scientists interested in the chemistry and biological actions of metal complexes,
to those physicians involved in treating diseases such as arthritis, gastrointest-
inal ulceration, inflammatory bowel diseases, and Alzheimer's and related
inflammatory/degenerative dementias.
We hope that this book will also serve as a stimulus for more, indeed much
needed, research into the development of novel metal therapies, drugs that alter
metal ion status, and the molecular and cellular biology underlying the actions
of copper and zinc and their metal complexes.
Our thanks go to Ms Nettie Dekker, Mr Phil Johnstone and their colleagues
at Kluwer Academic Publishers who have helped in putting this book together
and gave much valuable advice. We also thank Veronica Rainsford-Koechli and
Marguerite Lyons for their invaluable secretarial help and assistance in
coordinating the preparation of this book. This book is especially dedicated to
those scientists and physicians who have persevered in their understanding of
the actions and applications of metal ion therapies as well as the roles of copper
and zinc in various diseases.
(For the editors)

K.D. Rainsford
Sheffield Hallam University, Sheffield, UK
ix
1
History of zinc therapy
TU Hoogenraad
Department of Neurology, University Hospital, Utrecht, The
Netherlands
HISTORY OF ZINC THERAPY

Paracelsus
Theophrastus Bombastus von Hohenheim, 1493-1541, the Swiss physician who
called himself Paracelsus, was the first clinician in Europe to suggest the
prescription of chemical therapeutics in medicine (Figure 1). Paracelsus sharply
broke with traditional beliefs when he applied his interest in alchemy and metals
to the treatment of his patients. In so doing, he earned himself the title 'father of
pharmacology' [1]. Zinc sulphate (sal vitrioli) was part of his pharmaceutical
arsenal and was termed gi11a Theophrasti [2].
GAUBIUS
The first scientific paper on zinc therapy dates back to 1771 [3]. It was written in
Latin and titled 'Luna fixa Ludemanni'. The paper forms a chapter in the book
Adversariorum varii argumenti written by Gaubius (1705-1780) at the end of his
career as professor of medicine and chemistry at the University of Leiden.
Gaubius was one of the successors of Boerhaave, the renowned professor of
medicine in Leiden who had emphasised the importance of chemistry. In his
paper on zinc therapy Gaubius described how he discovered that a secret drug,
'luna fixa', sold by a renowned alchemist, Ludemann, consisted of nothing else
but zinc oxide [4]. "Memimeram . . . sanationes miraculosas, lunae fixae
tributas. Infantem ex diris convulsionibus desperatissime decumbentem, a
medico ordinario depositum, minima pulvisculi huius dosi aegerime ingesta
penitus liberatum quasi revixisse scio". ("I remembered miraculous healings
attributed to the use of luna fixa. I know of a child in a desperate condition,
confined to its bed with severe convulsions. The child was given up by the official
medical doctor. With great difficulty a miniscule dose of the powder was
administered after which the child was rescued and recovered.")
In the subsequent paragraphs, Gaubius described his studies of the effect of
zinc in the treatment of convulsions and spasms. He ended his article with the
K.D. Rainsford et a/. (eds.), Copper and Zinc in Inflammatory and Degenerative Diseases. 1-5.
© 1998 KJuwer Academic Publishers.
~.d.LT£lUVS NON SIT.QYl SVVS USE. POT.ESr.
>4hVl\£OLl THEOPHRASTI AS HOHE.NHAI M

£FflCJU SV~ li.TATIS +S·
AV. PH. TH. PUACSur. NATt AN. 149,. MOltTVL AN. ...st-
Ar. SV& ..7. r.u.
Figure 1 Paracelsus at the age of 45. Engraving by Augustin Hirschvogel, 1538
remark that it was too early to make statements about the indications and the
reliability of the drug. "Probatae fuerint fidei ... experientia duce constituator"
("the reliability of the agents should be established with experience as guide").
ZINC AS ANTIEPILEPTIC
In the 19th century oral zinc therapy was used in the treatment of epilepsy. Zinc
oxide was found to oe a relatively safe drug, even though the doses prescribed in
those days were extraordinarily high and were administered for long periods. To
give an impression of the high doses of zinc used: the psychiatrist Schroeder van
der Kolk of Utrecht recommended zinc oxide (ftores zinci) in a daily dose of
about 1800 mg elementary zinc [5], corresponding to a daily dose of 7800 mg
2
zinc sulphate. This is approximately 13 times the dose we prescribe nowadays

for many of our patients with Wilson's disease (l35 mg elementary zinc per day,
corresponding to 600 mg zinc sulphate). Herpin of Paris, who prescribed zinc
oxide for most of his patients with epilepsy sometimes used even higher doses
[6], one of his patients being treated with a daily dose of 4.800 mg elementary
zinc!
ZINC AS EMETIC AND ANTIDOTE FOR INTOXICATIONS

In the second half of the 19th century, interest in zinc therapy for epilepsy
declined so that, in the first part of the 20th century knowledge about its long-
term administration seemed to have been forgotten completely. Nevertheless,
because of its low toxicity and emetic effect, zinc sulphate was still included in
most European pharmacopeas as an emetic and antidote for intoxications [2].
ZINC TO TREAT COPPER TOXICOSIS IN WILSON'S DISEASE

In more recent years there has been a revival of interest in zinc therapy. In 1961,
Schouwink, a neurologist at the University of Amsterdam, wrote his thesis on
the influence of zinc supplementation in autosomal recessive copper toxicosis
(Wilson's disease) [7]. He had learned from the veterinary literature that high
doses of zinc sulphate could lead to decoppering in sheep [8]. Schouwink
performed copper balance studies in two patients with Wilson's disease and
could demonstrate that the copper balance became negative when zinc was
administered in a dosage of three times 45 mg per day. He prescribed zinc
sulphate as long-term treatment to the two patients. Many years later, the
disappearance of Kayser-Fleischer rings in one of these patients was proof that
effective decoppering of patients with Wilson's disease can be accomplished with
zinc therapy [9]. This finding, and knowledge that the use of zinc salts is not
accompanied by severe side effects, formed the basis of zinc therapy as
treatment for Wilson's disease [10,11].
ZINC THERAPY FOR ZINC DEFICIENCY SYNDROMES

In 1963 Prasad published an article on the effect of zinc supplementation in
zinc-deficiency syndrome [12]. The syndrome is characterized by growth
retardation, dwarfism, hypogonadism, and delayed sexual development. Hypo-
geusia, anorexia and impaired wound healing are other consequences of chronic
zinc deficiency. In these patients, zinc supplementation results in acceleration of
growth, normalization of sexual development, taste acuity and improved wound
healing.
3
ZINC AS A DRUG FOR ACRODERMATITIS ENTEROPATHICA

In 1973, Barnes and Moynahan reported on a baby wi th signs of acrodermatitis
enteropathica caused by a synthetic diet grossly deficient in zinc. The skin
lesions promptly disappeared when zinc supplements were added to the diet.
This observation led to the discovery that the clinical syndrome of acroderma-
titis enteropathica is due to zinc deficiency. The primary defect in this autosomal
recessive disease appears to lead to malabsorption of zinc [13]. In 1975, the
Committee on Drugs of the American Academy of Paediatrics recommended
that zinc should be the therapeutic agent of choice in newly diagnosed cases of
acrodermatitis enteropathica.
TOXICITY OF ZINC: COPPER-RESPONSIVE LEUCOPENIA AND

ANAEMIA
Herpin mentioned that most of his patients who recieved zinc oxide had only
minor side-effects, such as vomiting and gastrointestinal irritation and that
long-term treatment was without noxious side effects [6]. Although he reported
that one of his patients became very pale after a cure with zinc oxide, he did not
recognize that excessive doses of zinc induce anaemia. We now know that zinc,
given in high doses for long periods, induces copper deficiency. Copper-
responsive leucopenia and anaemia are the major signs of this deficiency.
In recent years, only a few noxious effects of zinc have been observed in
humans, apart from some incidental disorders due to an extremely high intake
of zinc, of the order of several grams of elemental zinc per day. Metal fume fever
represents a temporary reaction of some workers to inhalation of zinc oxide.
The acid fumes from zinc chloride in smoke bombs can produce irritation of the
eyes and airways.
In 1926, it was observed that zinc chloride injected into the testes of animals
induced cancer. However, as zinc salts are unlikely to be administered by
intratesticular injection, this finding has little relevance to human medicine [14].
CONCLUSION
In 1771, Gaubius introduced zinc into academic medicine with the words: I offer
you a medication with many promises. Since then more than two hundred years
have elapsed and some of these promises have come true. It is an active therapy
for two serious diseases and it is without major noxious effects. Nevertheless,
one should not be surprised if new possibilities for this remarkable drug are
soon found. For instance, the possibilities of using zinc for several inflammatory
diseases seem promising [15].
4
REFERENCES
1. Petrucelli R1. The renaissance. The fifteenth and sixteenth centuries. In: Lyons AS, Petrucelli
RJ, eds. Medicine, an Illustrated History. New York: Abrahams; 1978:376-7.
2. Pereira 1. De beginselen der materia medica en der therapie, voor Nederland bewerkt door
LCEE Fock. Amersfoort: WJ van Bommel van Vloten; 1849.
3. Gaubius HD. Adversariorum varii argumenti. Lunafixa Ludemanni. Leiden: Luchtmans;
1771.
4. Hoogenraad TU. Luna fixa Ludemanni or the introduction of oral zinc therapy in official
medicine. Trace Elem Med.l984;1:47-9.
5. Schroeder van der Kolk JLC. Bau und Functionen der Medulla spinalis und oblongata und
nachste Ursache und rationelle Behandlung der Epilepsie. Braunschweig: Vieweg und Sohn;
1859.
6. Herpin T. Du prognostic et du traitement curatif de l'epilepsie. Paris: JB Balliere; 1852.
7. Schouwink G. The continuing story of copper and zinc. In: Scheinberg IH, Walshe JM, eds.
Orphan Diseases and Orphan Drugs. Manchester: Manchester University Press; 1986:56-62.
8. Dick AT. Copper toxicosis in sheep [dissertation]. Melbourne: University of Melbourne;
1954.
9. Hoogenraad TU, Koevoet R, de Ruyter Korver EGWM. Oral zinc sulphate as long-term
treatment in Wilson's disease (hepatolenticular degeneration). Eur Neurol. 1979;18:205-11.
10. Hoogenraad TU, Van den Hamer CJ, Koevoet R, de Ruyter Korver EG. Oral zinc in Wilson's
disease [letter]. Lancet. 1978;2:1262-3.
II. Milanino R, Deganello A, Marrella M et al. Oral zinc as initial therapy in Wilson's disease:
two years of continuous treatment in a 10-year-old child. Acta Paediatr. 1992;81: 163-6.
12. Prasad AS. Deficiency of zinc in man and its toxicity. In: Prasad AS, Oberleas D, eds. Trace
Elements in Human Health and Disease. Vo1.2. New York: Academic Press; 1976:1-20.
13. Barnes PM, Moynahan EJ. Zinc deficiency in acrodermatitis enteropathica: multiple dietary
intolerance treated with synthetic diet. Proc R Soc Med. 1973;66:327-9.
14. Leonard A, Gerber GB. Toxicity of essential and beneficial metal ions: zinc. In: Berthon G,
ed. Handbook of Metal-Ligand Interactions in Biological Fluids. New York: Marcel Dekker;
1995:705-8.
IS. Rainsford KD, Whitehouse MW. Anti-ulcer activity of a slow-release zinc complex, zinc
monoglycerolate (Glyzinc). J Pharm Pharmacol. 1992;44:476-82.
5
2
Physiological properties of copper and zinc
V Albergoni
Department of Biology, University of Padova, via U. Bassi 58/B,
35121 Padova, Italy
INTRODUCTION
The properties of metals in biological compartments depend not only on their
characteristics, but also on those of the ligand sites which interact with them.
From this interaction derive properties and structural characters which confer a
functional role or roles on the biological molecules and compartments in which
the metals are present. The various distributions and locations of the ligands in
organs and structures may confer often very different but interacting roles on
the metals. Therefore, the physiological properties of metal ions, in this case
copper and zinc, must be described with reference to the systems in which they
are involved. Nor is it surprising that the natural or experimentally induced
depressed state of metals provides the first indications of their putative effects,
and data regarding functional roles are often closely linked and mingled with
those related to a deficient state. Trace elements such as copper and zinc have
important functions in both humans and animals, are known to be essential,
and must be available in adequate amounts depending on absorption,
distribution and storage in a regulated system which obviously includes
excretory mechanisms. These aspects therefore form the basis of all functional
roles and physiological properties.
ZINC
Absorption, transport, tissue distribution and excretion
Zinc is one of the most abundant trace elements. The total body content is about
20 g, 58 J..lg/g w/w in liver and 55 J..lg/g w/w in kidney. The normal human adult
requires an intake of about 15 mg zinc per day [1]. Absorption of dietary zinc
has been estimated at about 20-40% and depends on source; zinc is absorbed
better from animal sources than from vegetable ones, but recent data indicate
that cereal proteins enhance its absorption [2]. Many ligands, especially phytate
and fibres in general, bind zinc, like other metals, and render it unavailable for
absorption. Zinc is absorbed mainly as a complex in the small intestine [3,4]. In
fact, zinc ion is preferentially coordinated by many ligands; such complexes then
7
© 1998 Kluwer Academic Publishers.
occur at the surface of the intestinal mucosa. The enhancing or inhibitory effects
of various ligands in comparison with free ion, e.g. when ZnCh is used in
experimental animals, should be evaluated with caution. The role of glycocalyx
in mineral absorption may be inferred, although the precise mechanism is
unknown. A carrier-mediated process occurs across the brush border mem-
brane. No induction of a membrane carrier protein has been observed but Zn-
binding proteins of different molecular weight have been found.
The role of intracellular binding ligands in zinc absorption is debated, and a
key role for metallothioneins (MTs) in this process has been suggested, but
somewhat conflicting data are also reported regarding the general role of
endogenous ligands. In any case, zinc uptake depends on a non-saturable
transport process not affected by dietary zinc intake and a saturable one
stimulated by zinc depletion, depending on a carrier-mediated mechanism [5]. A
cysteine-rich intestinal protein which binds zinc during transmucosal transport,
not apparently influenced by zinc status, has recently been identified and isolated.
This is an 8.6-kDa protein with 7 cysteine residues, and its gene is not (or only
minimally) expressed in organs other than the small and large intestine [6].
Only a fraction of most trace elements in the human diet is absorbed, and this
fraction depends on a number of physiological factors so that correct evaluation
of intestinal processing of zinc, like that of other elements, is difficult and also
depends on evaluation of zinc turnover rate and loss [7]. All absorbed zinc must
pass via the plasma to other tissues and is estimated at 5 mg/day [8]. The
concentration in plasma (20% of whole blood) is about 15 JlmollL, nearly 84%
loosely bound to albumin which acts as the main carrier, 15% tightly bound to
an cx2-macroglobulin, and 1% ultrafilterable and mainly bound to amino acids.
Zinc content in erythrocytes is I mg/ 106 cells, whereas, in leucocytes, it is 6 mg/
106 [3,9]. Some zinc is bound to MTs present in plasma and blood cells.
The plasma zinc concentration, like its distribution between the extracellular
fluid pool and tissues, is closely associated with its intake in diet and is sensitive
to hormonal control (glucocorticoids, epinephrine, glucagon) and to various
stress conditions. Absorbed zinc is taken up by the liver, and this occurs rapidly.
The mechanism of zinc uptake by cells has been studied in rat cultured liver
cells. Its uptake in the presence and absence ofligands has been compared [10]
and this appears to be similar to that for intestinal absorption. Likewise, in
human erythrocytes, the presence of two carriers is strongly supported, one at
low and another at high affinity for zinc transport, the data indicating that the
zinc uptake from deficient cells is clearly increased and correlated with zinc
status [II]. Other data on endothelial cells indicate that zinc transport is an
energy-independent carrier-mediated process [12]. Recent data on in-vitro
endothelial cells supports the presence of two major pathways on zinc transport:
one involves a receptor-mediated saturable co-transport zinc-albumin by
transcytotic vesicles, the other involves non-saturable co-transport with ligands
(principally albumin and histidine) through intercellular junctions [13]. Other
data regarding the kinetic parameters of zinc transport in proximal cells isolated
from rabbit kidney cortex show that Zn may also enter complexed with cysteine
8
PHYSIOLOGICAL PROPERTIES OF COPPER AND ZINC
or histidine via a sodium/amino acid co-transport mechanism [14].

There is no specific zinc store although, according to the data reported by
some authors [15-17] a limited one may exist in bone. Zinc in bone does not
appear to be readily available for mobilization in various experimental condi-
tions. In any case, during bone reabsorption, deposited zinc is released, with
redistribution of the metal in response to its decreased supply [18]. Zinc content
and distribution in the organism is explained by the amount of the metal
required and present in biomolecules and biostructures for functional purposes.
Redistribution of zinc from plasma to liver also seems to depend on glucocorti-
coid hormones, epinephrine, glucagon and interleukin-l (IL-l); this effect has
been correlated with the enhanced synthesis of MTs induced by hormones.
Bacterial endotoxins also produce hypozincaemia, coupled with hypercuprae-
mia due to enhanced ceruloplasmin levels; this effect is mediated by IL-1 [9]. A
close inverse relationship has been described between hormonally regulated
hepatic metallothionein synthesis and serum zinc levels.
Like those of copper, the metabolic steps of zinc are influenced (as previously
noted) by the characteristics of the ligands and by their interactions with metals.
Metal ions entering the compartment may be bound, according to their
chemical characteristics and those of the binding group, first in a 'buffer system'
and then transferred to, or trapped by, functional subsystems (soluble or
structural components). They may also induce specific chelating molecules
involved in their storage or excretion [19]. Different zinc concentrations are also
maintained at the intracellular compartment level. In cytoplasm, it is < 10-9
mollL whereas, in many vesicles, it is > 10-3 mollL; a mechanism for moving
the metal by a pump has been hypothesized but not demonstrated [20]. In the
little-known equilibria between free and bound zinc ion and zinc metabolism, a
central role may certainly be ascribed to MTs. These low-molecular-weight
proteins contain a significant amount of zinc. Other metals, especially copper
in mammals, may also be bound. In fact, it should be noted that mammalian
MTs have a regulatory MT gene containing a metal-responsive element, a
steroid-responsive element, and a transcription factor [21], and that the amount
of MTs present in tissues mainly depends on zinc status [22].
Gastrointestinal excretion is the main excretory route, and is calculated as
2.5-5.5 mg/day; the renal route accounts for an excretion rate of 5-10 ~mollday.
Intestinal excretion, apart from the unabsorbed fraction, occurs either via cell
desquamation and transmucosal flux or, mainly, via digestive secretion, mainly
pancreatic; this means that excreted zinc is in any case bound to endogenous
ligands, of which MT is probably the main one [3,5]. Intestinal excretion
depends to some extent on nutritional zinc status, whereas renal excretion,
mostly by tubular secretion, remains fairly constant and is about 300--700 ~g/
day [23]. Hormonal regulation of renal zinc excretion is also reported: insulin
inhibits while glucagon increases excretion [9]. Maintenance of zinc homeo-
stasis is due to endogenous excretion rather than to the absorption mechanism.
Further zinc losses are due to sweat and skin desquamation, which certainly do
not involve regulatory mechanisms.
9
Functional roles
Zinc is required in nearly 300 enzymes, playing catalytic, co-catalytic and/or
structural roles for the proper folding of proteins. A structural role is also played
in RNA polymerases and Zn-proteins involved in nucleic acid replication. Zinc
ligands at the catalytic centre and regulatory sites in some enzymes have been
well characterized, together with their spatial arrangement, to give a convincing
description or indications regarding the relationships between structure and
function [21,24]. According to its presence in different biological compartments,
the functional role of zinc varies and is complex. Many data on these aspects are
presumed from Zn-deficient conditions in animals, and direct relationship to
that in humans is often lacking.
Most of the interesting data regarding the role of zinc in replication, transcrip-
tion and translation have been obtained from bacteria and unicellular eukaryotes.
A role in the proper folding of the DNA-binding domains oftranscription factors,
including hormone receptor proteins, has been demonstrated, and is strongly
supported also for zinc in DNA polymerase, DNA-dependent RNA polymerase,
and accessory Zn-proteins involved in nucleic acid replication. Zinc is also a
component in an ATPase stimulated in the presence of DNA, probably involved in
5S RNA gene transcription [21,24,25].
The role of zinc in protecting biological structures from damage by free
radicals may be due to several factors: maintaining an adequate level of MTs,
which are also free radical scavengers; as an essential component of superoxide
dismutase; and as a protective agent for thiols and other chemical groups, since
zinc prevents their interaction with iron and thus free radical formation [26].
Zinc deficiency increases the levels of peroxidated lipids in mitochondrial and
microsomal membranes, whereas its presence prevents lipid peroxidation by
membrane stabilization [27-29). Other data indicate that a low zinc content
increases the osmotic fragility of erythrocyte membranes, and that spectrin and
actin are more readily dephosphorylated. Zinc is an effector in tubulin
polymerization and acts in vitro on actin filament formation and stabilization;
the latter three proteins also bind zinc. Dietary zinc deficiency also alters
membrane skeleton protein composition reflected by adducin content and
another unidentified protein band [30]. The effects of zinc deficiency have also
been analysed in some physical parameters of erythrocyte 'ghosts'. Membrane
fluidity becomes greater as a function of this deficiency. ESR and spin label
probes suggest effects on the physical state of the lipid bilayer and cell surface
carbohydrates, and indicate a stabilizing effect of zinc [31). Experiments on
endothelial cell monolayers show that albumin transfer is depressed in a low
zinc medium [32].
The role of zinc as a component essential for the development and
maintenance of the immune function has been highlighted by severe zinc
deficiency in patients with differing pathologies, particularly in genetic disorders
of zinc metabolism. In these states, there is reduced absorption of this metal.
Primary or secondary zinc deficiency produces a decrease in T-cell number, and
10
the response of T-lymphocytes to phytomitogens, in T-cell-dependent antibody

production, and in NK-cell activity. An Ig-related receptor involved in
inhibition of NK cells, which requires zinc for its function, has recently been
described [33]. The reduced activity of thymic hormones has also been described
in zinc deficiency. An early effect of this condition is the reduction of tymulin
concentration in serum: it has been pointed out that tymulin, which requires
zinc for its function, is involved in many steps of the cell-mediated immune
responses. It should be noted here that zinc is the only naturally occurring
lymphocytic mitogen found in the body [34-36].
Another effect of zinc deficiency on blood cell components is impaired
haemostasis due to defective platelet aggregation. This has been demonstrated
in rats although the biochemical defect is still unknown [37].
The role of zinc in nervous functions has only received limited investigation.
Due to the high zinc content of the cortex and hippocampus, most detailed
studies have concentrated on the latter. Zinc is apparently concentrated in the
terminal axonal bags. Hippocampal slices have shown that electrical
stimulation increases the zinc turnover rate (in and out) in mossy-fibre
neurophil, suggesting that the metal plays a role in the neural signalling process
[38]. The effect of zinc on excitatory amino acid receptors has been studied in
cultured hippocampal neurons. These studies indicate an inhibitory effect on the
N-methyl-D -aspartate receptor channel and a modulatory effect on channel
opening [39]. Endogenous zinc may also modulate GABA-mediated synaptic
transmission, at least in the immature rat hippocampus [40], whereas
contradictory data have been reported for the postsynaptic effects of zinc on
GABA receptors. At the presynaptic level, zinc may act by blocking Ca2 +
channels, thus inhibiting neurotransmitter release, as demonstrated in single
calcium channels in mouse myotubes [41]. Zinc and enkephalin distribution are
identical in rat and guinea pig brain. Binding of some tested enkephalin to
opiate receptors in various areas of the brain is inhibited by a zinc concentration
range compatible with that in the same regions of the brain. It has also been
shown that the presence of zinc reduces receptor affinity by increasing the
dissociation constant of the receptor for naloxone. Zn inhibits binding of
ligands to each of the various classes of opioid receptors, although to different
extents [42--45]. These data, not comprising all studies, only emphasize the role
of zinc in synaptic transmission and neuroreceptor modulation.
With the aim of clarifying the role of hormones in trace-element metabolism,
hormonal deficiencies have been experimentally produced by gland ablation
and metal distribution has been analysed. Many of these hormonal deficiencies
induce marked changes although some of these are contradictory and permit
only limited evaluations of the specific role of the hormones involved in the
distribution of copper and zinc among tissues. In these studies, it is already
difficult to distinguish between primary and secondar effects. Only in a few cases
have other experimental approaches permitted more substantial information.
The most significant data concern the presence of zinc with regard to secreted
insulin, the role of glucocorticoids on zinc uptake and transport by mammary
11
glands [46], and the relationships between zinc and hormones involved in
growth and the reproductive cycle [47,48]. Abnormalities of skeletal growth are
reported in low zinc status, as well as pronounced hypogonadism in males [49].
Zinc deficiency leads to altered metabolism of androgens, oestrogens and
progesterone, prostaglandins and somatomedin C. The metal binds to peptide
hormones and thus confers proper configurations, or acts at the level of
membranous or nuclear receptors which, in the case of steroid receptors, are
zinc-finger proteins [47,48]. A recent extensive review of zinc biochemical and
physiological aspects has been published by Vallee and Falchuk [50], and
constitutes a useful integration of the data summarized here.
COPPER
Absorption, transport, tissue distribution and excretion
The total copper content in humans is about 90 mg, 20-30 times lower than
zinc, e.g. 5-6 ~g/g w/w in liver and 2 ~g/g w/w in kidney [51]. The normal adult
requires 2-3 mg Cu per day. Absorption of the metal in a normal diet has been
estimated at 30% of total intake. Copper absorption takes place from the
stomach to the middle part of the intestine. It is unlikely that copper remains
in its ionic form, but is readily complexed. In this respect, the stoichiometry of
binding, nature of the binding site and kinetic behaviour of the exchanges
between various ligands are all important. I-Amino acids facilitate copper
absorption more than d-isomers, and protein-mediated transport may also
occur. Zn, Cd, Hg and Ag interfere in absorption of Cu by competition for
binding sites and for effects in many metabolic systems. Fibre effects of copper
availability to humans is minimal [4]. Ascorbic acid reduces absorption and
sulphide blocks it, rendering the metal unavailable for the formation of Cu
complexes required for transport across the intestinal mucosa; leguminous
proteins also inhibit its absorption [2,4,52].
Copper is transported in blood as Cu2 +, mainly bound to I-histidine; it also
forms several ternary complexes in which albumin may be involved. The
fraction bound to amino acids may facilitate rapid transport to tissues. A role
of ceruloplasmin as a copper transporter is highly probable. A reduction step
may occur, with subsequent metal trapping by a Cu + acceptor [52-54]. Copper
derived from ceruloplasmin enters the cell but the protein does not; the effects of
several chelators indicate that copper is taken as Cu+ rather than Cu 2 +. Other
experimental data indicating the presence of mediated Cu-transport are in
favour of the presence of a ceruloplasmin receptor, and many putative receptors
have also been identified [55]. Free Cu uptake is another common mechanism,
and has been characterized as an energy-independent saturable, probably
carrier-mediated, process. The presence of various ligands has different effects
[55]. Data from several types confirm the presence of a common mechanism
operating like the intestinal one. Antagonistic interactions with other metal ions
show that the carrier is not highly specific.
12
Copper enters the liver from plasma, and at least half of the ceruloplasmin
copper appears in vesicles; from these, it is transferred to newly synthesized Cu
proteins. A copper fraction may be tightly bound to MTs and taken up by
lysosomes prior to excretion into the biliary canaliculi. Copper content in the
liver is directly related to intake, and the metal is present in all subcellular
fractions. Aside from its role in the synthesis of Cu proteins, the liver is the
principal site of storage and excretion, but the regulatory mechanism of the
intracellular processing of copper has not yet been completely clarified. The
main steps of the process include uptake, intracellular distribution and
utilization, and export. On the basis of genetic studies of unicellular organisms,
a P-type ATPase, active in Cu export from the cell, is highly probable in
humans, and data on Wilson's and Menkes' diseases indirectly support this
hypothesis [55]. A bombesin-like neuropeptide, neuromedin C, has been
indicated in the transport of Cu2 + within the nervous system. Cu2 + may interfere
with neurotransmission or growth factor effects ofneuromedin C [56].
As for copper metabolism, a central role is ascribed to MTs which, together
with glutathione have a main role in metal detoxification. In any case, a Zn-
dependent increase in MT content and in the copper bound to MTs by
displacing zinc, causes inhibition of copper uptake and may increase the
amount of excreted metal [51,55,57]. Copper distribution in various cellular
compartments and its delivery to Cu proteins are poorly understood [55]. As for
control mechanisms, it is reported that epinephrine and glucagon stimulate
copper incorporation into ceruloplasmin, while glucocorticoids and sex hor-
mones increase ceruloplasmin secretion from hepatocytes [58].
In adult man, about 2 mg of Cu are absorbed in the intestine and excreted in
the bile each day. Bile is the main route for copper excretion, as it contains low-
molecular-weight binding components (amino acids and small peptides) as well
as high-molecular-weight species, probably secreted and found prevalently in the
gall bladder complexed with various biliary components, and the metal is not
available for reabsorption. Copper excretion may occur from lysosomal
exocytosis, as deduced from data on Long-Evans Cinnamon (LEC) rats
[51,53,59]. Urinary excretion is negligible, since few Cu compounds, mainly
amino acids and MTs, permeate the glomerular filter, and their total or partial
reabsorption has not been verified. The kidney also releases a small percentage
of copper into urine [52,58].
Functional roles
Copper enzymes and non-enzymatic Cu proteins are widely distributed; the
specific effects of the metal in some 100 Cu enzymes that involve a wide range of
activity have two peculiar functions: electron transport, and O2 transport and
metabolism. Another very important role of copper is in iron mobilization and
absorption, in which ceruloplasmin may function as ferroxidase catalysing the
oxidation of Fe2 +, and thus probably loading of Fe3 + onto transferrin and
apoferritin. Ceruloplasmin also plays a role as an antioxidant, possibly related
13
to its free-radical scavenging properties in extracellular compartments.

Scavenging activity in intracellular compartments is performed by the Cu-Zn
enzyme SOD [60]. Copper deficiency in rats significantly reduces SOD activity
in aorta, associated with a decrease in prostacyclin production and an increase
in lipid peroxidation [61]. In this respect, it has also been found that lipoproteic
fractions from copper-deficient rats are more susceptible to oxidative damage,
with lowered SOD, catalase and glutathione peroxidase activities [62]. Copper
deficiency affects plasma lipid composition, mainly by increasing the amount of
total cholesterol and plasma triglycerides, which seem to be sustained by
enhanced synthesis and secretion instead of reduced elimination [62,63].
Many other data on the roles played by copper come from nutritional copper
depletion studies in humans and animals and from pathological conditions.
Many diseases are in fact associated with modifications of copper content and
distribution, although there are as yet no further or convincing data to explain
the mechanisms involved in these disorders. Copper deficiency is responsible for
defects in bone growth and repair, probably due to the role of the metal in cross-
linking of bone collagen. For cardiovascular integrity too, particularly that of
major arteries, normal cross-linking of elastin and collagen is important [64,65].
Copper levels are also related to female gonadal function, and the metal appears
to be involved in the action of estradiol [49]. Another interesting effect of copper
deprivation is related to immunocompetence. Impairment of immune functions
occurs in severe copper deficiency: in particular, reduced lymphocyte
phytoemoagglutinin and concanavalin A stimulation indices, a stable specific
antibody response, a reduced IgM concentration [66], and also a reduced
capacity to respond to the T-cell-independent antigen S3 [67] have been
observed in rats. Copper also seems to be required for a variety of other
functions: proper cardiac function, connective tissue development, mye-
linization of the spinal cord, keratinization, and tissue pigmentation [68].
CONCLUSIONS
Although only representing a small part of the literature on this topic, the
information reported here allow some reflections and evaluations to be made.
The different distribution of specific metals primarily occurs according to the
genetically determined distribution of their ligands, and their extent in relation
to the phases of the life cycle. As variations in metal content in tissues are
determined by metal-ligand affinity, including here the various transport
systems or ionic channels, they depend on all the possible interferences on the
same site; in this respect, the interactions of essential elements and toxic metals
are particularly important. Copper and zinc participate together in many
common physiological systems or share some functional aspects, including:
(a) The presence of active zinc transport in entering cells and putative copper
active export from cells may indicate a common homeostatic mechanism;
(b) Both metals are present in metallothioneins, as barriers for intestine-
14
blood transport, as mechanisms for the probable regulation of intra-

cellular metal contents, and in any case for heavy metal detoxification
(the data discussed here, integrated with other aspects and a global
evaluation of the functional role of Cu-Zn MTs, are treated in another
chapter of this book);
(c) Cu and Zn are also both present in SOD, which acts as a free scavenger,
and plays a role in lipid peroxidation; and
(d) Their deficiency alters immunocompetence.
The role of zinc in biological systems seems to be more varied and is better
documented than that of copper, but Cu-Zn interactions and their regulation
certainly deserve deeper study, including more complete evaluation of patholo-
gical conditions.
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17
3
Biological chemistry of copper compounds
L-O Klotz and U Weser
Physiologisch-chemisches Institut der Eberhard-Karls-Universitat,
Anorganische Biochemie, Hoppe-Seyler Strasse 4, TObingen,
Germany
Copper is well known as a biochemically essential transition element. Attribu-

table to both its kinetic and redox activities, it plays a key role in electron
transfer reactions. In-viv0 oxygen chemistry is dependent on and regulated by
copper proteins: all reduction states of dioxygen are connected with copper
proteins and related copper compounds (Figure I).
The subject of this article will be the biological chemistry of low-molecular-
weight copper compounds. Copper proteins - most of those found in mamma-
lians and characterized to date are listed in Table 1 - will not be explained in
detail.
COPPER IN ANCIENT MEDICINE

Until the dawn of the age of antibiotics, copper-containing mixtures and drugs
were of great importance for the treatment of inflammatory diseases. Our
knowledge about the use of copper in medicine reaches back to the Middle
Kingdom of ancient Egypt. The Papyrus Ebers originating from that era
(approximately 1550 BC) contains a summary of recipes for medically used
ointments or suspensions based on copper compounds [1,2]. Similar
preparations used in ancient Roman medicine are described by Pliny the Elder
(23-79 AD) in his Naturalis Historia (P.E., nat. hist., [3]). Copper ores were
processed in many different ways which may seem rather curious, but which
resulted, as we now know, in copper compounds which indeed have their own
pharmacological reactivity, such as copper oxides, copper sulphide, copper
sulphate, malachite (Cu(OHh.CuC0 3) and verdigris (copper acetates; the
production of verdigris to be used as paint pigment following medieval recipes
that resemble the former ancient instructions mainly yielded Cu(CH3 COOh.
[Cu(OHh]4.3H20 = 'basic cupric acetate (143)' [4]). Two examples may be
highlighted: the use of copper sulphide (sory, misy; P.E., nat. hist. XXX and
XXXI [3]) for the 'treatment of the eyes' and against tonsilitis (P.E., nat. hist.
XXX and XXXI) must essentially be attributed to its low solubility resulting in
19
K.D. Rainsford et al. (eds.), Copper and Zinc in Inflammatory and Degenerative Diseases. 19-46.
REDUCTION STATE OF RELEVANCE OF COPPER

DlOXYGEN
O 2 (dioxygen) - dioxygen transport in

molluscs and arthropods
(hemocyanin)
- substrate for copper
dependent oxygenations (e.g.
tyrosinase, dopamine p
monooxygenase)
- substrate for a multitude of
copper-dependent oxidases
O 2-. (superoxide) - superoxide dismutation

(Cu,Zn-superoxide
l+e- /2W dismutase, copper complexes)
H20 2 (hydrogen peroxide) - product of copper-

dependent superoxide
dismutation
- product of oxidations
catalyzed by "non-blue"
oxidases (e.g. galactose
oxidase, copper-dependent
amine oxidase)
HO· (hydroxyl radical) - product of hydrogen

peroxide reduction via
copper-dependent Fenton
reactions
- product of a variety of
copper-dependent oxidations
(e.g. cytochrome oxidase,
"blue" oxidases)
Figure 1 The roles of copper in oxygen biochemistry. In biological systems, any reduction state of
molecular oxygen is linked to copper proteins or low-molecular-mass copper compounds
a constant release of low concentrations of pharmacologically reactive copper

ions [5]. Verdigris mixed with honey was successfully applied against tonsilitis
(P.E., nat. hist. XXV). In fact, this preparation should be very effective since
cupric acetate exerts strong anti-inflammatory activity [6]. Compounds of the
Cu-acetate type effectively catalyse superoxide dismutation in vitro [7). Super-
oxide radicals are generated in the course of inflammatory processes. In
20
BIOLOGICAL CHEMISTRY OF COPPER COMPOUNDS
addition, hydroxyl radicals produced as by-products in both copper-dependent

Fenton chemistry and during inflammation are thought to be scavenged by the
honey's polyol content.
PARAMETERS USED TO EVALUATE THE BIOCHEMICAL ACTIVITY OF

COPPER COMPLEXES
Copper complexes synthesized for one of a diversity of reasons (cf. pp. 26ft) will
- like any substance designed for such a purpose - have to be categorized with
regard to some parameters if their special usefulness is to be evaluated. Some of
the important categories are listed below.
Catalytic activity
Most low-molecular-weight copper complexes, if their co-ordinative features
allow the binding of superoxide to the metal centre, have superoxide dismutase
(SOD) activity, i.e. they catalyse the dismutation of superoxide according to:
SOD-active substances accelerate the decay of pulse-radiolytically generated

superoxide. They also inhibit the reduction of dyes such as nitro blue tetra-
zolium chloride by superoxide formed in vitro, e.g. by the xanthine/xanthine
oxidase system. For extensive summaries and discussions of both direct and
indirect SOD assays [see 8,9]. In addition to superoxide dismutase activity and a
series of other catalytic properties (cf. pp. 26ft), copper compounds have been
tested for catalase activity [10,11], for the catalysis of oxidative cleavage of
nucleic acids (as with 'chemical nucleases', like Cu(I)-orthophenanthroline [12-
14] and for hydrolytic activity (including hydrolytic cleavage of nucleic acids
[15-17], partly even in a site-specific manner, e.g. by synthetic ribozymes [18]).
It is of the utmost importance to elucidate whether or not a specific catalytic
activity will be exerted in vivo and, if so, under what conditions.
Stability
Knowledge of the stability and intactness of copper complexes is indispensable
for evaluating their biochemical fate. Complexes administered to biological
systems are influenced by the presence of many a biomolecule that is a potent
copper chelator. There may be, for instance, constituents of cell culture media
or, on a physiological scale, of the blood and extracellular fluids that impair the
availability of copper to cells by simply being more efficient ligands than the
original ones and thereby forming (e.g. macromolecular) complexes that cannot
be transported across physiological barriers like membranes.
Mammalian serum contains copper in a concentration of roughly 15 IlmollL,
90-93% of which are bound to ceruloplasmin [7,19]. About 1 IlmollL (7-10%)
21
Table 1 Mammalian copper proteins*
Molecular Copper
Protein weight (kDa) content Function References
8
Amine oxidase 2x92 2 x 1 Cu (type II) Oxidative deamination of primary amines (histamine, 126--128 ~;c
(copper-containing putrescine, and others): »
EC 1.4.3.6 RCH2NH2+H20+02- RCHO+ NH3+ HzO z z
o
N
Z
Lysyl oxidase 1 x 32 1 Cu (type II) Oxidation of peptidyl-Iysine in elastin and collagen to amino- 129 o
EC 104.3.13 adipic semialdehyde (cross-linking of protein fibre is initiated) Z
Z
Cytochrome c oxidase 205 3 Cu ([CU21A, CUB) Mitochondrial terminal oxidase of cellular respiration 130--133 "
EC 1.9.3.1 (13 subunits) catalysing the reduction of dioxygen to water using ~
s::
electrons of ferro-cytochrome c
o~
~
Dopamine 13- 2 or 4 x 72.5 2 or 4 x2 Cu Catalyses a key step in the biosynthesis of catecholamines: 134--137
'"'"
mono-oxygenase (type II) 3,4-dihydroxy-phenylethylamine+02+ascorbate
~
o
EC 1.14.17.1 -noradrenaline+H 20+dehydroascorbate o
!Hm
Peptidylglycine 1 x 38-75 2 Cu (type II) Processing of glycine-extended precursor-peptides to 138-141 z
m
mono-oxygenase yield C-terminally amidated bioactive peptides:
(peptidylglycine cx- (a) Peptidylglycine cx-hydroxylating mono-oxygenase
~
amidating enzyme) activity (EC 1.14.17.3): peptidylglycine+2 ascorbate+
iii
o
EC 1.14.17.3 02-peptidyl-(2-hydroxy)glycine+2 semidehydro-
ascorbate+H 20 ~
m
(b) Peptidylamidoglycolate-Iyase activity (EC 4.3.2.5): C/J
peptidyl-(2-hydroxy)glycine-peptidylamide+
glyoxylate
Table 1 (cont)
Molecular Copper
Protein weight (kDa) content Function References
Monophenol 1 x 55-70 2 Cu (type III) Key enzyme in melanine biosynthesis; catalysed reactions: 142-144
mono-oxygenase (a) L -Tyr+L -dopa +0 2 ..... L-dopa +dopaquinone+ H 2 O
!!l
(= tyrosinase) (b) Monophenol+0 2 ..... 0-diphenol+H 20 (cresolase activity) 0
EC 1.14.18.1 (c) 20-diphenol+02 ..... 20-quinone+2H2 0 (catecholase activity) 6G>
Cu,Zn-superoxide 2x 16 2 x 1 Cu (type II) Catalysis of superoxide dismutation; 9,103,105, ~
r'
(')
dismutase and and and singlet oxygen decontamination; 145-149 :r
extracellular (EC-) 4x 30 (EC) 4 x 1 Cu (type II) copper stress protein m
s:
superoxide dismutase
EC 1.15.1.1 ~
'<
J'I.)
W 0
'T\
Ceruloplasmin 1 )( 132 6Cu (a) Principal copper transporter in human plasma; 150-151 (')
0
EC 1.16.3.1 (3 x type 1; 1 x type II; (b) FerrOltidase activity: 4Fe(II)+4H+ +0 2 ..... 4Fe(III)+ 2H 2 O "IJ
"IJ
(ferroxidase) 2)( type III) m
::0
Metallothionein 1 x6.8 Cu(n-thiolate clusters Metal (including copper) ion homeostasis; scavenging 152-154 8s:
"IJ
(up to 12 Cu(I» of reactive oxygen species 0
C
z
Neurocuprein lx9 1 Cu (type II) Regulator of dopamine (3-monooxygenase? 155-156
c(J)
Factor V (proaccelerin) 1 x 330 1 Cu (type II) Blood clotting 157
Factor VIII 300 (80+220) 1 Cu (type I) Blood clotting 177
·For additional listing of non-mammalian copper proteins, see References 158 and 159
protein
specific metal binding
human SA Asp ala - his - Iys - yes

rat SA Glu ala - his - Iys - yes
bovine SA Asp Ihr - his - Iys - yes
pig SA Asp Ihr - Iyr -Iys - no
dog SA Glu ala - Iyr -Iys - no
chicken SA Asp ala - glu - his- no
Figure 2 N-terminal copper-binding site of human serum albumin. The involvement of Lys-4 in
copper binding was proposed by Sadler et al. [167]. The alignment of the four N-terminal amino
acids of various serum albumins (SA) [168] demonstrates the necessity of Ris-3 for specific copper
binding: human, rat and bovine SA do bind copper(II) specifically; pig, dog and chicken SA do not
of copper is found coordinated to albumin (serum concentration about 700

~mollL [7]) [7,19]. Low-molecular-weight complexes - most likely employing
histidine (serum concentration approximately 130 ~mollL, all amino acids
totalling 40 mmollL [7]) - constitute a minor residual fraction (2-4 nmollL
[7]). More recent publications show 'transcuprein' to be an additional blood
copper transporter; the following copper distributions are found: 62.5%-71 %
copper bound to ceruloplasmin, the rest being associated with serum albumin
(15.6-19%), transcuprein (7-12.5%) and present as low-molecular-mass (low
Mr) complexes (2-9.4%) [20,21]. Most mammalian serum albumins have an N-
terminal-specific copper-binding site (Figure 2) capable of forming stable
complexes (log Kcu(II)-BSA = 16.2 [22]). Together with the unspecifically ion-
binding proteins, peptides and amino acids, they form the blood's battery of
chelators that has to be faced and overcome by an administered copper co-
ordination compound.
24
In order to elucidate whether or not a copper complex is stable under

competitive chelating conditions, one can investigate mixtures by electron
paramagnetic resonance (EPR) which is suitable for the detection of changes
in typical copper(II) signals. Their hyperfine structure [22-24] is indicative of
alterations in the copper co-ordination sphere. Sometimes, the maintenance of a
catalytic activity (such as SOD activity) is a parameter for the stability of a
complex [25]. In the case of copper-diSchiff-base complexes, circular dichroism
studies were employed to estimate stability constants: the formation of Cu(II)-
serum albumin from BSA in the presence of varying concentrations of the low
Mr complexes tested was visualized using the characteristic negative Cotton
band of Cu(II)-BSA in the 550 nm [22] to 564 nm [25] region. The low Mr
compounds exhibited no chiroptic properties in the same spectral region [22,25].
Following the shifts in d-d-absorption maxima and the rise or decay of copper
thiolate luminescence [26-28] are two of many other means to follow structural
changes in copper co-ordination compounds.
Redox behaviour
Experiments describing the redox properties of copper compounds (e.g. cyclic
voltametry, reduction kinetics, etc.) are of utmost importance for gaining insight
into their biochemical reactivity. Under conditions of extracellular matrix, i.e. in
extracellular fluids like serum, copper should be most stable in its cupric form
(Cu(II)). By way of contrast, due to the reducing conditions inside the cell,
intracellular copper is mainly present as Cu(I), Cu(II)-CuZn-SOD being a
remarkable exception. Intracellular reduction of copper compounds proceeds
mainly via oxidation ofthiols [29,30], the most prominent being glutathione. In
biological systems, copper compounds can also be reduced by ascorbate [30--32]
or hydrogen peroxide [33]; electron transport chains have also been discussed as
copper reductants [34]. A reduction by NADH or NADPH is neither found in
the case of the Cu-diSchiff-base complex Cu-PuPy* [26] nor of Cu(II)-
phenanthroline [35]. The reduction of cupric 1,1O-phenanthroline depends on
the presence of NADH but proceeds indirectly via an NAD radical and only if
H 2 0 2 is available [36]. In the presence of oxygen, intracellularly formed Cu(I)
species can lead to damage to many a biomolecule [37-39]. Generally, the
hydroxyl radical HO" is considered to be the ultimate oxidant during these
processes and is believed to be generated in a Fenton-like reaction sequence
(reactions (1)+(2b)). Yet, the estimated steady-state concentrations of oxygen
and hydrogen peroxide under physiological conditions are about 10-5 mollL
and 10-9 mollL, respectively [40]. As a consequence, the production of hydroxyl
radicals should proceed in hypoxic situations only; otherwise, redox cycling
according to reactions (l )+(2a) should be favoured:
*[N,N'-bis(2-pyridyl-methylene)-1 ,4-butanediamine]-(N,N' ,N",N"')-copper(II)
25
2R-SH+2Cu2 + -+ 2Cu+ +R-SS-R+2H+ (1)

2Cu ++ 2H+ +0 2 -+ 2Cu2 ++ H 2 0 2 (2a)
Cu++H2 0 2 -+ Cu 2 ++OH-+HO' (2b)
Degradation of H 2 0 2 can be catalysed by copper compounds £10,11]. This

decomposition advances via reduction of copper to Cu(I) which, in turn, can
react with another molecule of H 20 2 according to reaction (2b). Therefore, this
'catalytic' activity of some copper compounds is of no pharmaceutical interest.
By way of contrast, the superoxide dismutating activity of a series of copper co-
ordination compounds can be pharmacologically exploited (cf. pp. 26ff, 30ft).
Mechanistically, this reaction depends on the reversible reduction of the
cuprous centre. Another prerequisite for SOD activity is the accessibility of the
central ion to reductants or an easily replaceable ligand so that superoxide can
interact with the copper centre.
Rather different from the enzyme, there is no substrate-guiding protein
moiety surrounding the active site. Thus, the specificity of complexes displaying
SOD activity for superoxide is limited. This is valid especially inside cells where
there are reductants other than superoxide (estimated steady-state concentra-
tion 10-11 mollL [40]) in much higher concentrations, such as glutathione
(present in the 1 mmol/L range [41], up to 19.2 mmollL in nuclei [32,42]).
Glutathione will preferably reduce cupric complexes intracellularly. Accord-
ingly, no intracellular SOD activity exerted by low Mr complexes will be found;
there will rather be redox cycling at the expense of GSH and O 2 (reactions
(l)+(2a».
Li pophilicity
Knowledge about the partition behaviour of copper compounds in biphasic
systems (such as octanollbuffer [43,44], liposome/buffer [30,43,44] or cellular
systems [43]) helps to estimate the distribution of these substances in physiolo-
gical conditions.
SELECTED COPPER COMPOUNDS

Most copper complexes are investigated and partly even designed for one of
three reasons. Some serve as models for the in-vivo fate of copper. Hence,
biologically relevant ligands are used to synthesize copper complexes: sugars
[45,46], sugar derivatives (like heparin [47]), amino acids [7,48-52], peptides
[7,49,52-59] (including peptides serving as models for the N-terminal copper-
binding site of serum albumin, Gly-Gly-His [60,61] and Asp-Ala-His [62]), and
others (e.g. nucleotides [63,64], phytates [65]). Some of these compounds were
found to exert catalytic activities, mostly superoxide dismutating activity [7,48-
51,53,56,57]. Regarding the stability constants of the respective copper co-
ordination compounds (Table 2), the in-vivo significance of this remains
26
Table 2 Stability constants of Cu(II)-complexes
Ligand /OglO K/* References
Formate 1.57 0.65 7

Acetate 1.67 0.98 7
Salicylate 10.45 18.47 7
Glycine 8.38 6.87 78
Histidine 10.2 18.5 7, 51
10.3 7.62 78
Histamine 9.58 6.48 78
Cimetidine 4.48 4.37 78
Uracil 4.55 64
Hypoxanthine 6.0 64
Gly-gly-his 12.8 78
Asp-ala-his-NMA t 16.96 62
TAABt 18.06 160
Pulmt 17.1 22
PuPyt 16.1 22
PuPhePyt 18.33 25
BSAt 16.2 7,22
HSAt 16.18 37
*K. =[CuL]/[Cu] [L]; K2 =[CuLiJ/[Cu] [Ll'; L =ligand

tAbbreviations: NMA: N-methylamide; TAAB: tetra-anhydroaminobenzaldehyde; Pulm: 1,8-di(2-imidazoyl)-
2, 7-diazoocta-l, 7-diene; PuPy: N,N' -bis(2-pyridyl-methylene)-1 ,4-butanediamine; PuPhePy: N,N'-bis(2-pyri-
dyl-phenyl-methylene)-1,4-butanediamine; BSA: bovine serum albumin; HSA: human serum albumin
questionable. Furthermore, copper complexes are designed as structural models

of copper proteins in order to elucidate catalytic mechanisms. Examples include
certain models for the oxygen-transporting protein, haemocyanin [66-68],
models for blue copper proteins [69,70], substances exhibiting copper-depen-
dent mono-oxygenase activities (oxidative hydroxylation [67,68,71] and meth-
oxylation [71], oxidative dealkylation [71-73] and dehalogenation [71]), models
for copper thiolate clusters in biological macromolecules (e.g. of copper
thionein or the copper-dependent transcriptional activator, ACE1) [74]. Lately,
nitrosyl copper complexes serving as paradigms for the copper-mediated
reduction of nitrite to nitric oxide and the further disproportionation of NO to
NOi and NzO by nitrite reductases were designed [75,76]. In fact, those
compounds do exert catalytic activity, but this is not the main aim, for, in
mechanistic studies, a catalytically active complex with a turnover rate which is
not too high should be most useful because kinetic studies would then be
facilitated. The third category of copper compounds is synthesized for their
possible catalytic activity which is to be utilized and exploited for pharmaceu-
tical or medicinal purposes.
It has been proven for many drugs that co-ordination with copper intensifies
their pharmacological reactivities and/or diminishes side-effects [6]. Antiulcer
27
agents, such as the H 2 -receptor antagonists, cimetidine and famotidine, are

fairly potent copper chelators [77-80]. The binding of cimetidine to imidazole
receptors in rat brain is increased in the presence of cupric ions [77];
additionally, Cu(I)-cimetidine and Cu(II)--cimetidine are superoxide dismutat-
ing substances [78]. Because of the low stability of the Cu(II) form
(log K = 4.48), it is thought to play only a minor role in vivo [78]. The main
degradation product, cimetidine sui phoxide, binds copper even less [80]. By way
of contrast, Cu(I)-cimetidine is rather stable (log K =9.11 [78]) and its
formation perhaps is the reason for the effects of copper on, the pharmaco-
dynamics of cimetidine.
Omitting additional examples [81-85] - a very extensive discussion of this
matter by Sorenson [see 86] - the following short section will deal with the very
thoroughly examined copper complexes of non-steroidal anti-inflammatory
drugs (NSAIDs). It has long been known that, in inflammatory diseases such
as rheumatoid arthritis, there is an increase of serum copper levels [86].
Although the reason for this phenomenon is unknown, one can speculate that
it results in copper becoming more available, rendering the formation oflow Mr
copper complexes feasible employing ligands found in blood. As a huge variety
of Cu low Mr compounds was found to exert superoxide dismutase activity,
including copper-amino acids and copper-peptides [7], a role in the scavenging
of reactive oxygen species may be presumed, produced by polymorphonuclear
leucocytes, such as superoxide, hydrogen peroxide, hydroxyl radicals, singlet
oxygen, hypochlorite and nitric oxide [87].
Similarly, the in-vivo formation of copper-NSAIDs was postulated to be the
true cause of their anti-inflammatory activity which, in fact, was found to be
intensified in the presence of copper ions [6,86,88]. Although, for instance, the
formation of low concentrations of Cu-indomethacin in serum was proven
experimentally [89], the hypothesis becomes very unlikely, regarding the low
stability 'of Cu-NSAID complexes (Table 2). In an in-vitro assay employing
EDTA as a model for physiological copper chelators [90], it was shown that Cu-
NSAIDs of the acetate type (Figure 3) are very unlikely to show superoxide
dismutase activity in vivo. On the contrary, the SOD-like activities of copper
complexes of antiarthritic agents, such as penicillamine and related compounds,
were rather stable in the presence of surplus EDTA.
Copper must, therefore, modulate the pharmacological reactivity of NSAIDs
in a different way not yet elucidated.
In their anti-inflammatory activity, NSAIDs depend on [91]: (a) their
extensive binding to serum albumin (!), (b) the maintenance of their hydro-
philic-lipophilic polarity, and (c) their pK values (around 4) which must not
change significantly. In addition, the pharmacokinetics of NSAIDs are quite
interesting [91]: although no receptors are known and although arachidonate
metabolites (the production of which is impaired by the NSAID-dependent
inhibition of cyclo-oxygenases [92-94]) are fairly ubiquitous, a regional accu-
mulation is found; gastric mucosa, kidneys, liver, blood, bone marrow, and the
sites of inflammation contain large amounts of ingested NSAIDs [91]. Copper
28
R=
-H (Formate)
-CH, (Acetate)
HO
--b (Salicylate)
'---CH,
H,CWOCH' (Indomethacin)
I
o"c~
~CI
6 (Lonazolac)
~,~-6v
Figure 3 Antiferromagnetically coupled and EPR-silent acetate-like Cu(I1) complexes. Lipophili-
city and thermodynamic stability of the CU2Ligand4-dusters increase from top to bottom. Three
non-steroidal anti-inflammatory drugs (salicylates, indomethacin, lonacolac) are chosen as ligands
[7,169]
could modulate the action of NSAIDs by interfering with the distributing

processes needed for proper anti-inflammatory action: although this seems to
be a very simple model (perhaps too simple), it is not unreasonable to propose a
Cu2+ -mediated binding of NSAIDs to serum albumin. The NSAID-carbox-
ylate moieties could bind to a Cu2 + fixed to nitrogen donors on the protein's
surface - it is known, for example, that acetylsalicylate acid interacts with Lys-
199, undergoing a transacetylation reaction [95]. This would yield a ternary
complex which may be stabilized by the additional interaction of the lipophilic
moieties with hydrophobic crevices of the protein. In regions with lowered pH,
such as the points of inflammation, the co-ordination features of this complex
might be changed. The respective NSAID molecule is released and can diffuse to
its site of action. This model could also be applied to drugs acting on specific
receptors (e.g. Hrantagonists; see above): copper could mediate serum-protein-
dependent transport to the point of action which, in this case, is the respective
receptor that can be presumed to bind to the drug much more strongly than the
protein--copper moiety. In this way, undesired reactions of drugs due to unco-
ordinated distribution could be minimized in the presence of copper.
A major disadvantage of NSAIDs is their detrimental effects on the gastric
mucosa. Recently, NO-releasing derivatives (nitroxybutylesters) of flurbiprofen,
29
ketoprofen and diclofenac have been designed in order to achieve a site-specific

NO release which stimulates gastric mucosal blood flow, concomitantly
counteracting the effects of cyclo-oxygenase inhibition [92]. Serum-stable super-
oxide dismutase active copper complexes (see below) are presumed to support
this effect by prolonging the life time of NO [96] (Figure 5).
BIOCHEMISTRY OF COPPER COMPOUNDS: AN EXAMPLE

The life time of cytosolic Cu,Zn-superoxide dismutase in blo~d is limited by
proteolytic degradation and is of the order of minutes [57]. The fairly large
molecular size does not allow simple diffusion across biomembranes. Thus,
many substances, most of which, in analogy to the active centre of CuZn-SOD,
contain copper, that exhibit SOD activity have been synthesized in order to
obtain a tool for transferring the beneficial effects of external CuZn-SOD, such
as anti-inflammatory activity [97-99], into a cell. Unlike the intact enzyme, the
use of these mimics allows a systemic application, avoiding an undesired
immunological response.
Early SOD-mimicking substances of the biuret or acetate type (cf. pp. 26ft)
are of pronounced superoxide dismutating activity if measured in systems free
from competing chelators [7,49,50,100], just like Cu 2 + .aq itself [49]. Their
stability under physiological conditions, however, is limited (Table 2). Addition-
ally, the intermediately formed soft Cu(I) in the course of superoxide dismuta-
tion cannot be stabilized properly by the hard oxygen donors.
For this reason, copper-diSchiff-base complexes were designed as Cu,Zn-
superoxide dismutase analogues [22,25,101,102]. Four nitrogen atoms co-
ordinating the central copper in an open-ring manner simulate the Cu,Zn-
SOD situation: four soft histidine-imidazole-nitrogens surround the active
site's copper atom [103] as is necessary for the activity of the enzyme, whereas
the zinc found bridged to the copper via an imidazolate has both stabilizing and
supporting functions [104]. The 5,7,5-ring complexes are synthesized from
putrescine (I,4-butanediamine) and imidazole-2 aldehyde and pyridine alde-
hyde derivatives, respecitvely (Figure 4), in the presence of copper salts (cupric
perchlorate is preferred for the perchlorate's crystallization properties). The
putrescine bridge forming a seven-ring system together with the chelated copper
facilitates a rapid change in the copper's oxidation state. It enables the complex
to rapidly change from square planar (Cu(lI) to tetrahedral (Cu(I» co-
ordination - the way it is thought to be in Cu,Zn-SOD [103] (recent findings
modify this view [l05]). Contrary to acetate- or biuret-type complexes, copper-
diSchiff-base complexes have extraordinary stability against chelators encoun-
tered in biological systems (Table 2). Thus, their SOD-like reactivity survives the
presence of excess BSA or EDTA [22,25]: a lO-fold (Cu-PuPy) to 100-fold
(Cu-PuPhePy*) molar excess of EDTA is needed to seriously impair the SOD
*[N,N'-bis(2-pyridyl-phenyl-methylene)-1 ,4-butanediaminej-(N,N',N", N"')-copper(II)
30
2 2
R 0
+ Cu(elO,),
("'l in EtOH
NH, NH,
pH 5-6
2 H2 O
(C10 4 12
OVo
I,
rl
N/ 'N
I R = H: Cu-PuPy
R = C.Hs: cu-PuPhePy
Figure 4 Scheme describing the synthesis of Cu-diSchiff bases from pyridine-2-aldehyde (R =H;
Cu-PuPy) or 2-benzoylpyridine (R = C6HS; Cu-PuPhePy) and putrescine (1,4-diaminobutane).
For Cu-Pulm, imidazole-2-aldehyde is taken instead ofpyridine-2-aldehyde [22,25,101,102,170]
activity of the complexes (measured under these conditions the superoxide

dismutating properties of Cu(II)-EDTA [106] are negligible (pH 8». Rate
constants for superoxide dismutation by diverse copper compounds were
determined by following the decay of pulse radiolytically produced superoxide
at 245-250 nm (Table 4). Interestingly, the rate constants of Cu2+ (aq) and some
acetate-type copper complexes (no competing chelators present) exceed those of
CU,Zn-SOD and the diSchifI-base complexes. Obviously, the axial lahn-Teller-
distorted H 2 0 ligands can be exchanged more readily in this type of complex
than in the enzyme and diSchifIbases. Yet the physiological significance of these
values is more than questionable. The superoxide concentrations used in these
experiments are unphysiologically high, and ubiquitously present biological
chelators strongly impair these ideal dismutation activities simply by competing
for the copper ion of the complexes and forming copper-ligand adducts with
lower reactivities. This ligand exchange, however, does not take place with the
intact enzyme and copper-diSchifI bases. Thus, the latter should be able to
maintain their superoxide dismutating abilities under (non-reducing; see below)
physiological conditions.
By scavenging superoxide radicals, SOD and stable analogues prolong the life
time of the physiologically important mediator, nitric oxide (NO), which
31
nitroxyl anion NO-2 + 102
CU(I)-PuPy
or Cu(l)-SOD
1 Cu(II)-PuPy
... or Cu(II)-SOD
NO + O~ ONOO- ---~ N03'
I
peroxynitrite
scavenging of superoxide
by SOD-active substance
ONOOH
I
Figure 5 The fate of nitric oxide in biological systems depends on the presence of superoxide. NO
produced from L-arginine or pharmaceutical NO-donors can either be reduced to the nitroxyl
anion reversibly [108] or directly degraded to peroxynitrite during the reaction with superoxide
(k2 = 5.6 x 10 7 moIlL- 1 S-I; 37°C [171]). This reaction can be hampered by scavenging superoxide
via superoxide dismutation [172]. Peroxynitrite can either decompose to nitrate or form hydroxyl
and N02 radicals [173-175]. These we1\-known reactive species are able to damage biomolecules.
Alternatively, singlet oxygen and nitrite are proposed to result from a reaction of peroxynitrite
with hydrogen peroxide [176], the steady-state concentration of which was estimated to be about I
nmollL under physiological conditions [40]
otherwise would decompose to nitrate via peroxinitrite (ONOO"), a very reactive

and neurotoxic [107] species (Figure 5). The degradation of NO released from
pharmacologically interesting donors (including glyceryl trinitrate) is delayed
thereby, and its diffusion radii should be increased_
Cu(II)-SOD reversibly reduces NO to the nitroxyl anion, NO-, which does
not react with superoxide [108]. The presence of such a pre-equilibrium restricts
both the availability of NO and its decay. The reversible reduction of NO is also
catalysed by Cu(l)-PuPy [96] which, in this respect, resembles the enzyme_
In analogy to CU,Zn-SOD, the copper centre of Cu-diSchiff bases is co-
ordinated to four nitrogen atoms: the enzyme's four histidine imidazoles are
replaced by two imino and two pyridine (Cu-PuPy and Cu-PuPhePy) or
imidazole nitrogens (Cu-Pulm*). The catalytic properties improve from Cu-
*[1 ,8-di(2-imidazoyl)-2, 7-diazooctadiene-l, 7]-(N, N',N", N"')-copper(JI)
32
Table 3 Superoxide dismutase activities of copper(U) complexes*
Amount of Cur II)

for 50% inhibition
ofNBTreduction
Compound (lJmoIIL) References Commentst
CU,Zn-SOD 0.04 22, 101, 102, 161, 163 1; pH 7.8

0.0084 25 1; 2; ph 8.0
0.0041 162 I; 8; pH 7.8
0.003 56 2; 3; pH 7.8
Cu-Pulm 4.0 22, 102 I; pH 7.4
Cu-PuPy 1.4 22,101,102 I; pH 7.4
0.6; 0.839; 0.995 25 I; 2; 6; pH 8.0
Cu-PuPhePy 0.27 25 I; 2; pH 8.0
Cu-TAAB 0.144 162 1; 7; 8; pH 7.8
Cu-EDTA 52.0 106 l;pH7.5
17.5 106 1; pH 7.0
Cu(salicylateh 16.0 161 1; pH 7.8
Cu(acetylsalicylateh 23.0 161 1; pH 7.8
Cu(di-isopropylsalicylateh 73.0 161 1; pH 7.8
Cu(lysh 86.0 22, 101, 102 I; pH 7.4
Cu(tyrh 45.0 101 1; pH 7.4
Cu[cyc1o(Hishh 0.03 56 2; 3; pH 7.8
Cu,Zn(im)-L 0.5 163 I; 4; 5
'Measured as copper concentration needed for 50% inhibition of NBT reduction by superoxide; NBT =nitro
blue tetrazolium chloride. In order to compare genuine SOD activities, one would have to compare the activity
values of the compounds related to the activities of the intact enzyme; for example, the IC so values of Cu-TAAB
and Cu-PuPhePy are 0.144 Ilmol/L and 0.27 IlmollL, respectively. Yet, under the given experimental
conditions, both complexes show 3% of the enzyme's activity.
tComments:
I. The xanthine/xanthine oxidase system was used to generate superoxide
2. Measured in the presence of competing EDTA
3. K0 2 dissolved in DMSO, was used as the source of superoxide
4. Measured in the presence of bovine serum albumin
5. Imidazole-bridged heterobinuc1ear CU,Zn complex of a macrobicyclic ligand
6. Activities measured I h, 12 hand 7 d after dissolving in H 20
7. TAAB =tetra-anhydroaminobenzaldehyde (macrocyc1ic ligand)
8. Iodophenyl-nitrophenyl-phenyltetrazolium salt (INT) was used instead ofNBT
Pulm to Cu-PuPy to Cu-PuPhePy (Table 3). The imidazole nitrogen in Cu-

Pulm is rather electron rich, whereas the pyridine-N in Cu-PuPy is fairly
electron deficient. An additionally electron-drawing phenyl residue in Cu-
PuPhePy increases the delocalization of the nitrogen electrons and the polariz-
ability of the pyridyl-to-imino-nitrogen system. As a consequence of this, the
capacity of the ligand to stabilize the soft Cu(I) transiently formed during the
superoxide dismutating process is improved.
33
Table 4 Second-order rate constants for the dismutation of pulse-radiolytically generated superoxide
by various copper compounds
Rate constant k2
Cu complex (moIlLs- 1 ) Reference Comments
Spontaneous dismutation 5.05 x 105 164 pH 7.2; I7°C

4.47 x 105 25 pH 7; 20°C
1.78 x 105 100 pH7
Cu2+(aq) 2.7 x 109 49
CU,Zn-SOD 3.1 x 109 164 pH 7.1; 37.5°C
2.0 x 109 164 pH 7.1; 20°C
I.3 x 109 49
Cu-PuPy I.I x 109 164 pH 7.1; 37.5°C
0.6 x 109 164 pH 7.1; 20°C
1.58 x 106 25 pH 7.0; 20°C; EDTA:Cu=4:1
Cu-PuPhePy 0.48 x 109 25 pH 7.0; 20°C; EDTA:Cu=4:1
Cu-TAAB 0.29 x 109 160
Cu(1ysh 0.56 x 109 49
Cu(tyrh 1.0 x 109 50
Cu(gly-hish 0.29 x 109 49
Cu(gly-his-Ieuh 0.21 x 109 49
Cu(formateh 2.7 x 109 7 pH 7.5
Cu(salicylateh 1.64 x 109 165 pH 7.5
Cu(acetylsalicylateh 0.96 x 109 165 pH 7.5
Cu(di-isopropylsalicylateh 2.4 x 109 165 pH 7.5
CU2(indomethacin)4 3.0 x 109 7 pH 7.5
Cu(indomethacinh 6.0 x 109 100 pH 6.6
Cu-penicillamine 0.4 x 109 7,166 pH 7.5
0.68 x 109 100 pH 7.0
Cu(I)-thionein 5.35 x 107 153 pH 7.2; Irc
TAAB =tetra-anhydroaminobenzaldehyde
The structural, spectroscopic and catalytic properties of CuPuPhePy (Tables

4 and 5) render it an extraordinarily good CU,Zn-SOD analogue, even better
than Cu-PuPy and Cu-Pulm.
In-vivo experiments revealed an anticarcinogenic effect of Cu-PuPy adminis-
tered intravenously in a rat carcinosarcoma model [109]; its general anti-
inflammatory efficacy was proven in male Wistar rats that suffered from
inflammation induced by potassium peroxochromate [110,111]. Moreover,
unlike Cu,Zn-SOD which has to be injected intra-articularly to exert antirheu-
matic effects [97], Cu-PuPy may be applied systemically to show antiarthritic
and anti-inflammatory activities [112]. Experiments performed in vitro regard-
ing the biochemistry of diSchiff-base complexes were carried out in a variety of
cell-free buffered systems [25,26,30,43]. A wealth of different cell types, e.g.
erythroleukaemia cells and human lymphocytes [26,30,113], Chinese hamster
34
Table 5 Magnetic behaviour and electronic absorption of copper-diSchiff bases and Cu,Zn-SOD
(data from References 22, 25 and 102)
Cu-Pulm Cu-PuPy Cu-PuPhePy CU,Zn-SOD
Visible electronic absorption (d-d transition)

Amax (nm) 690 710 690 680
E (mollL- 1 em-I) 35 116 136 150
EPR parameters
g.l 2.047 2.050 2.048 2.083
gil 2.234 2.225 2.217 2.271
All x 10-4 (em-I) 166 149 146 140
gil/Ali (em)* 135 149 152 162
*gll'All is an empirical parameter for the degree of tetrahedral distortion of a tetragonally co-ordinated complex
[25]. A value between 105 and 135 em is assigned to a square planar environment of the Cu(II} centre. A rise up
to 250 em is indicative of the distortion of the tetragonal co-ordination geometry against tetrahedral. The value
of Cu-PuPhePy is closest to that of CU,Zn-SOD
ovary cells [114,115], activated polymorphonuclear leucocytes [116], and rat

primary hepatocytes [43], were used.
Due to the stability of Cu-PuPy and Cu-PuPhePy, these complexes survive
proteinaceous and low Mr chelators found in blood. Additionally, it can
reasonably be assumed that no pronounced immunological response to the
application of Cu-PuPy or Cu-PuPhePy takes place. The membrane perme-
ability of Cu-PuPy was proven using liposomas [30]; owing to its phenyl
residues, Cu-PuPhePy has a greater lipophilicity (as reflected by octanollbuffer
and butanol/water partition coefficients; Table 6) and also permeates biomem-
branes, as was shown in liposomal systems and rat hepatocyte cultures [43].
Once migrated into the cell, copper-diSchiff bases are reduced and seem to
undergo redox cycling (reactions (3) to (5)). This was demonstrated for Cu-
PuPy, which is not reduced by NAD(P)H but by cellular thiols, the most
prominent of which is glutathione [26,30].
2 GSH+2Cu(II)-PuPy --+ 2Cu(I)-PuPy+2H+ +GSSG (3)

2Cu(I)-PuPy+02+2H+ --+ 2Cu(II)-PuPy+H 20 2 (4)
The net reaction is:
(5)
Redox cycling makes cells which are deficient in enzymes detoxifying hydrogen
peroxide (like many tumour cells [113,117]) prone to hydrogen peroxide-
mediated destruction. In contrast, a rise in glutathione peroxidase activity has
35
Table 6 Octanollbulfer* and butanollbulfer* partition coefficients of copper-diSchilf bases
Cu-complex Kp (octanollbuffer)t Kp (butanollbuffer)t
Cu-PuPhePy 0.7 ±0.31 ** 8.8 ±4.85

Cu-PuPy 0.14±0.06 0.22±0.21
'Phospate-buffered saline, pH 7.4, was used as the buffered aqueous phase

tKp =[compound]non_aqueous phase I (compound]aqueous phase
• 'Values are means of three independent measurements ± standard deviations
been found in a copper-resistant hepatoma cell line on treatment with copper

[118], possibly enabling the cells to effectively deal with an increased hydrogen
peroxide flux caused by elevated concentrations of cytoplasmic copper.
Lowering the concentration of intracellular GSH by incubating cells with
buthionine sulphoximine [26] or diethylmaleate [43] results in an elevated
sensitivity of the respectively tested cells to Cu-PuPy [26] and Cu-PuPhePy
[43]. Both complexes were shown to be reduced in the presence of the
intracellular milieux of ruptured cells [30,43] by following the decay of the
typical copper(II) electron paramagnetic resonance signal. This reduction could
be simulated in mixtures of copper-diSchiffbase plus GSH [26,43]. Cu(I)-GSH
was formed in the course of reduction and it was visualized by luminescence
measurements using the copper-thiolate chromophore as a marker [26,43].
Integrating both the risen toxicity of the copper complexes in GSH-deficient
cells and the fact that GSH participates in reducing copper intracellularly,
thereby mediating and causing its toxicity, a model is proposed for the
intracellular fate of Cu-PuPy [26] and Cu-PuPhePy [43]: glutathione reduces
intracellular copper(II) to Cu(I) and glutathione disulphide (GSSG). If GSH is
present in amounts high enough, it forms the redox-inert [119] Cu(I)-GSH that
functions as a mediator of copper transport into apo-copper-proteins [120-
123], concomitantly detoxifying copper by transferring it to metallothionein
(MT) - a process for which an intermediate ternary complex of MT, Cu(l) and
GS- has been proposed [118].
The model is consistent with the observation that the toxicity of cumene
hydroperoxide to rat hepatocytes is increased by Cu-PuPhePy in a concentra-
tion-dependent manner. This effect is strongly potentiated by the simultaneous
lowering of the cellular glutathione level to about 16% of its initial state by
preincubation of the cells with diethylmaleate [43]. The ability to transport
copper into cells perhaps is one of the reasons for cytotoxicity of copper-
diSchiff bases. In addition to the above mentioned effects, copper can be
liberated during reduction and inhibit specific proteins by blocking sensitive
dithiols. A prominent example for the inhibition by copper is glutathione
reductase [114,124] which is known to have a cystine in its active centre that is
transiently reduced to a dithiol in the course of the catalytic process [125].
36
PAP
ADP-ribosylation of NAD
ATP
mcr-
~ADP
I ~( 2 Cu(ID-dSb
'OH + OH- ";--~2GSH;ENADP ) OJ
,"HzO z H 20z o
Fe GPx GRed NRS 5Gl
" O2 2 H 20
HzOz
" ~ 2 Cu(D-dSb GSSG NADPH ~
()
., I
V-- GSH VProt-SH m
;::
,p.dSb I ";GSH
VJ
-...J Cu(D-GSH
~
o"T'I
export prot-SSG ()
o"U
"U
m
;0
1
transport of Cu(I) ()
o
into apo-copper-proteins ;::
"U
o
C
Z
o
(fl
Figure 6 Scheme illustrating the intracellular reactions of copper-diSchiff bases adapted from [26,30,43,114,115]. Hydrogen peroxide is detoxified by GPx
employing GSH. Concomitantly, oxidation and loss of pyridine dinucleotides, lowering of adenylate energy charge, oxidation and depletion (export of GSSG) of
glutathione take place. By way of contrast, Cu-diSchiff bases produce hydrogen peroxide in a redox cycling at the expense of GSH and O 2 . Because of the
inhibition of GRed by copper, GSSG accumulates, resulting in pronounced formation of prot-SSG and a lowered need for NADPH. PAP may be subject to such
a mixed disulphide formation and be inhibited thereby. Consequently, in H 2 0 2 stress produced by Cu-dSb, NADP(H) and NAD(H) redox ratios and adenylate
energy charge remain constant. H 2 0 2 may further be reduced by Cu(I)-dSb in a Fenton-like reaction to produce the extremely reactive hydroxyl radical. With
sufficient concentrations of GSH, Cu(I)-GSH is found as a beneficial chaperon for intracellular copper and, at the same time, no HO' is formed.
Cu-dSb, Cu-diSchiff base; FC, Fenton chemistry; GPx, glutathione peroxidase; GRed, glutathione reductase; GSH, reduced form of glutathione; GSSG,
glutathione disulphide; NK, NAD kinase; PAP, poly(ADP-ribose) polymerase; prot-SH, protein thiol (cys); prot-SSG, protein-glutathione mixed disulphide
As Nagele points out [114], this very inhibitory effect on glutathione

reductase by Cu-PuPy could be advantageous in the long run. Treatment of
Chinese hamster ovary cells with toxic doses of Cu-PuPy does not coincide with
typical effects elicited by hydrogen peroxide stress, although intracellular redox
cycling ofthe copper-diSchiffbase with glutathione and oxygen produces H 2 0 2 .
Both the inhibition of glutathione reductase resulting in the accumulation of
GSSG and the consecutively increased formation of mixed protein-glutathione
disulphides are proposed to be the basis reason. Poly (ADP-ribose) polymerase
(EC 2.4.2.30) is subject to such a 'glutathionylation' modification and is
inhibited thereby - with the consequences that adenyl ate pool, adenylate energy
charge and nicotinamide-dinucleotide redox ratios remain essentially un-
changed (Figure 6). Detoxification of intracellular copper via Cu(I)-GSH and
scavenging H 2 0 2 produced in redox cycling depend on glutathione status and
the presence of enzymatic defence mechanisms against oxidative stress. This
renders copper complexes of the diSchiff base kind selectively toxic, for many
tumour cells are deficient both in glutathione and in the appropriate enzymatic
scavenging machinery [26,30,113,117].
Apart from the possible therapeutic benefits of copper-diSchiff bases,
attributable to both their stability under non-reducing conditions and their
membrane permeability, they are an excellent means of studying the biochem-
istry of intracellular copper in cell culture.
ACKNOWLEDGEMENTS
We are indepted to Badreddin Abolmaali, Dr Jorg Muller and Dr Hans-Jurgen
Hartmann for stimulating discussions and help. Further thanks go to Regine
Muller for proofreading the manuscript. Part of this work was supported by
DFG grant We 401/24-3 and the Fonds der Chemischen Industrie.
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46
4
The biological chemistry of zinc
S Rahuel-Clennont and MF Dunn
Department of Biochemistry, University of California at Riverside,
Riverside, CA, USA
INTRODUCTION: ZINC IN BIOLOGICAL SPACE AND TIME

With about 3 g of zinc in the adult human body, zinc is second to iron in the
total mass of the trace elements in human biology [1]. Nevertheless, with the
isolation and identification of carbonic anhydrase as a zinc enzyme, its
biochemical importance was only recognized in 1939 [2]. The delay in the
development of the bioinorganic chemistry of zinc as a field, compared with
iron and copper, is attributable to its spectroscopic 'silence'. While complexes of
most transition elements are strongly coloured, the electronic structure of Zn2+
renders it colourless and this ion is not amenable to investigation by most
spectroscopic methods. This limitation has been overcome by the development
of ultrasensitive methods for detection of zinc (with a lower limit of 10-13 g)
based on atomic spectroscopy [3] and by techniques of structural biology for the
determination of the relationship between macromolecular structure and
function such as X-ray crystallography and NMR [3].
Zinc in biological space

The biological essentiality of zinc iron is revealed by the universal impairment of
cell division, growth, and differentiation under conditions of zinc deprivation
[3]. In a multicellular organism, virtually all zinc (> 95%) is intracellular, where
it is the most common trace element [1,3]; about 30-40% is located in the
nucleus, 50% in the cytoplasm, organelles, and specialized vesicles (e.g. for
digestive enzymes or hormone storage), and the remainder in the cell membrane
or wall [3]. The extracellular fraction includes plasma zinc bound to albumin
and zinc associated with secreted digestive enzymes (e.g. in the extracellular
matrix; see below), or with filamentous structures such as keratin [4,5]. Zinc ion
exists primarily in the form of complexes with proteins and nucleic acids, and
has been shown to be essential to the structure and function of a large number of
macromolecular structures including enzymes from all six classes, particularly
hydrolases [1,3]. Consequently, zinc ion participates in all aspects of intermedi-
ary metabolism, the transmission and the regulation of the expression of genetic
47
information [6,7], the storage, synthesis and action of peptide hormones [8-11],
and the structural maintenance of chromatin [3,12], biomembranes [13] and
extracellular matrixes.
The selection of zinc during evolution

Although adjacent elements in the periodic table usually display similar
chemical properties, living organisms take advantage of a wide range of
chemical elements, each of which have been selected for specific functions. The
average concentration of zinc ion in the human body ('" 10-3 moIlL), its
intracellular localization and its widespread biological functions imply active
selection and concentration processes which extract zinc from the pool
originally available in sea water ( '" 10-9 moIlL). The evolutionary scheme for
zinc selection proposed by Williams [14] gives an interesting prequel to the
unique properties of zinc ion detailed in the next section. Upon the change in
Earth atmosphere from a reducing H 2S/S composition to an oxidizing H 20/02
composition, zinc and copper would have become available from sea water in
the form of soluble hydroxide complexes, whereas previously they remained
inaccessible as very stable sulphide precipitates. Protein thiolates could then be
stabilized in the oxidizing extracellular environment by copper ion, the
strongest interactant, while zinc ion was selected by intracellular macromole-
cules in a still reducing medium. Interestingly enough, archaebacteria living in
niches removed from atmospheric oxygen still rely on cobalt and nickel
metalloenzymes.
In aerobic organisms, a complete set of specific chemical properties determine
the structure-function relationships between zinc ion and macromolecules. In
the following sections, we shall analyse the interplay of electronic and structural
interactions that convey to zinc its essential functions.
BIOINORGANIC CHEMISTRY OF ZINC

Zinc is the last member of the first row transition elements (in the fourth period
of the periodic table). The electronic configurations of these elements are
characterized by the progressive filling of the 3d atomic orbitals by up to 10
electrons. Therefore, Zn 2+ displays the advantageous reactivity and complexa-
tion properties shared by other transition metal ions, such as Mn2+, C0 2+, Fe 2+,
or Cu2+. However, the electronic structure of Zn 2+, [Ar]3d i04so, with the largest
atomic number Z of 30, provides this ion with the most versatile and malleable
co-ordination chemistry, resulting in a broad range of structural, catalytic and
regulatory functions [3].
Redox status
The ionization of Zno ([Ar]3di04s2) to the Zn2+ state occurs through removal of
the two electrons from the 4s shell. Compared with the preceding transition
48
THE BIOLOGICAL CHEMISTRY OF ZINC
elements, Zn (with nuclear charge Z =30) has a relatively high (unfavourable)

ionization energy for removal of the first electron, whereas loss of the remaining
s electron is comparable in energy to that of Fe, Co and Ni. On the other hand,
the removal of any additional electron from the filled 3d shell is highly
unfavourable. As a consequence, the Zn(lI) oxidation state is very stable
[15,16], neither the +1 nor the +111 oxidation states are accessible under
physiological redox conditions.
This redox stability renders Zn2 + suitable for interaction with nucleic acids by
avoiding the risk of corruption of the genetic material through free-radical
reactions [7,17]. Although Zn2 + is involved in the active sites of several
oxidoreductases, it does not act as an electron carrier as do the redox couples
Cu(I)/Cu(lI) in superoxide dismutase and Fe(II)/Fe(I1I) in cytochromes.
Complexation competence
Because the 3d shell electrons do not shield each other efficiently from the
nuclear charge, as the atomic number Z increases across the fourth period, each
individual electron is subject to an increasingly important attraction from the
nucleus. This results in smaller ionic radii [18] (Table 1) and, at equivalent
charge, a higher charge density, and a higher electrostatic potential around the
atom. Therefore, the ions of the last elements of the first transition row are
especially good electrophilic centres which seek the possibility of electrostatic or
covalent interactions to achieve charge neutralization [19,20]. This propensity to
form complexes generally follows the decrease of the ionic radii, as revealed by
the experimental stability series:
and is generally independent of the nature of the ligand [15,20].
Table 1 Selected ionic radii for tetraco-ordinate complexes 118)
Ion Ionic radius (AJ
K+ 1.51
Ca2+ 1.14 (hexaco-ordinate)
Mn2+ 0.80
Fe2+ 0.77
Co 2 + 0.72
Ni2+ 0.69
Cu 2 + 0.71
Zn 2 + 0.74
49
Thus, biological systems have evolved to employ the abundant, large, small-
charge metal ions (Na+,K+) in charge carrier functions, the large high-charge
Ca2+ ion in trigger and regulatory functions, whereas the transition metal ions
are involved in catalytic and structural roles [14].
The flexible nature of the Zn co-ordinate bonds

When the binding of a ligand to a metal ion involves the sharing of a lone pair of
electrons derived from the ligand, a co-ordinate bond is formed. The co-
ordination number of a metal ion complex identifies the number of co-
ordinating ligand molecules around the metal, which make up the ligand-field
of the complex. In accepting electron density from an electronic pair, the metal
ion acts as a Lewis acid. The reactivity of the co-ordinate bond depends to a
large extent on the ionic or covalent nature of the bonding. The greater the
electron transfer from the ligand to the metal, the more covalent the bond. This
transfer depends strongly on the tightness with which nuclei hold on to their
electrons (i.e. the polarizability) for both interacting partners [19]. Pearson
introduced the concept of hard and soft Lewis acids and bases [21] to describe
the co-ordination preferences of non-polarizable (hard) Lewis acids for non-
polarizable (hard) bases, leading to predominantly ionic bonding. Due to the
better mixing of the interactant deformable electron orbitals, low-charge large-
radius polarizable (soft) acids prefer polarizable (soft) bases, leading to bonds
with a higher degree of covalency. Examples of hard and soft Lewis acids
include the K+, Ca2+ and Mn2+ ions, and the Cu+, Cd2+ and Hg2+ ions,
respectively.
Zn2+ is a borderline acid, showing properties intermediate between hard and
soft. Therefore, it is able to form stable complexes with a wide array of bases of
variable softness. In biological and proteic environments, these bases include the
hard oxygen ligands from Asp and Glu carboxylate side-chains, carbonyl
groups, phosphoryl groups, and H 20, the borderline nitrogen ligand from His
side-chains, and the soft sulphur ligand from the Cys side-chain [19]. Within
°
proteins, amino-acid side-chains and water molecules are oriented to optimize
the alignment between the or N electronic lone-pairs and the Zn2+ ion [22],
indicating some covalency in these co-ordinate bonds.
Important functional consequences arise from the partially covalent nature of
Zn2+ co-ordinate bonds, which, moreover, can be modulated by the nature of
the ligands [19]. Indeed, Zn 2+ has the ability to polarize its bound ligands,
thereby opening the possibility for the activation of a water molecule or a
substrate within an enzyme active site (see below). Furthermore, due to its
relatively deformable 3d shell, Zn2+ is able to form additional stabilizing
covalent 1t-bonds through donation of 3d valence electrons to suitable ligands
such as Cys thiolate, a ligand found in both catalytic and structural Zn-sites of
enzymes (e.g. alcohol dehydrogenase) and other proteins with various functions
(see below).
50
The dynamic co-ordination geometry of Zn

Because of the shapes and orientations of the five 3d valence orbitals, each
ligand approaching a transition series metal ion in a particular geometry, e.g.
octahedral, tetrahedral or pentaco-ordinate, does not experience an equivalent
electronic environment. This non-equivalence gives rise to the stabilization of
certain orbitals relative to the others through splitting of the energy levels of the
3d orbitals. The associated stabilization energy depends on the number and
distribution of electrons filling the 3d orbitals and on the co-ordination
geometry. This ligand-jield effect is responsible for the stereochemical preference
of most transition metal ions for octahedral sites over tetrahedral sites
[15,16,19]. Nevertheless, in proteins, most Zn sites comprised of amino-acid
side-chains appear to be tetraco-ordinate [19,22,23]. Because the Zn 2 + 3d shell is
filled, this ion has a spherically symmetrical charge distribution, and hence is
not subject to ligand field effects upon switching from the octahedral solvated
free state to the tetraco-ordinate protein-bound state [24]. Furthermore, for the
same reason, the co-ordination geometry of Zn2 + in proteins is flexible. As a
consequeunce, Zn 2 + co-ordination geometry is adaptable to the constraints
imposed by the protein-derived ligand-field [25], which is frequently distorted
from the regular tetrahedral geometry in enzyme active sites; additionally,
flexible co-ordination geometry can favour enzymatic catalysis by easily switch-
ing from tetra- to penta- or hexaco-ordinate upon binding of substrates/
products and formation of intermediates (see last section).
The absence of ligand-field effects also results in the relative kinetic lability of
zinc co-ordinate bonds, as revealed by a higher rate of inner-sphere water
exchange (",20 IlS- 1), compared with other transition metal ions (only Cu2 + is
faster) [26]. Therefore, zinc ion has the ability to form stable and flexible protein
complexes which allow rapid ligand exchange dynamics, a combination highly
favourable for (a) the efficiency of enzymatic catalysis (see below), and (b)
regulatory purposes by means of promotion of protein folding (e.g. zinc-fingers
[24]), proenzyme activation (for the matrix metalloproteases [27], see below), or
protein-protein interactions [11,28].
Let us now turn to an analysis of the special properties of macromolecules,
particularly proteins, that allow zinc to carry out specific catalytic, structural
and regulatory functions.
ZINC CO-ORDINATION AND FUNCTION IN PROTEINS

The special nature of proteins as ligand
In addition to the stabilizing co-ordinate interactions described above, the
binding of Zn2 + to a protein appears to be a process driven by the large entropy
gain associated with the release of water molecules from the solvated ('free')
metal ion and the ('empty') protein site (see [22]). Indeed, this favourable effect
is not completely offset by the loss of protein conformational entropy upon
reorganization of the binding site. For many large proteins, the metal sites are
51
preformed [25]. In some systems, an unfolded smaller domain or a peptide

undergoes reorganization to give the metal-bound state [29]. In this case,
however, the metal ligands are borne by the same chain and therefore are
subject to an entropy loss upon binding that is smaller than that which would
have been experienced by the isolated ligands. The metal chelation provides the
energetic term necessary to overcome the unfavourable decrease of conforma-
tional entropy and allows the stabilization of a well-defined folded state [24].
Such interactions open the possibility of regulatory mechanisms in which, owing
to the fast kinetics of exchange of Zn 2 +, the binding of the metal triggers the
folding of the protein into its functional conformation.
Interactions at a protein metal site are not limited to the inner-sphere ligand-
field but include interactions from elements of the outer-sphere (i.e. the residues
and non-protein cofactors surrounding the metal complex) and distant elements
of the entire protein structure (see [22] and references therein). The affinity of the
site (its basicity towards the metal ion) has been shown to be affected by long-
range electrostatic effects, including the protein surface charge, localized (X-helix
macrodipoles, and direct H-bond networks involving zinc ligands. Owing to the
structure and H-bond donor nature of the His imidazolyl ring, this ligand is
susceptible to pKa modulation, as exemplified by the carboxylate-His-Zn
'triad' found in carboxypeptidase A [22,30] (Table 2, Figure 1). Such interaction
not only improves Zn 2 + complexation by enhancing the basicity and orienting
the His ligand, it also contributes to the reactivity of the zinc-bound water and
substrate within an enzyme active site. Similarly, a phosphate-His-Zn network
probably contributes to the recognition of nucleic acids by zinc-fingers [31].
Generally, the direct or indirect hydrogen bonding between inner-sphere ligands
and outer-sphere residues provides mechanisms for the promotion of nucleo-
philic reactions, the induction of the proper orientation of reactants, the
enhancement of water and ligand nUcleophilicity, and the stabilization of
intermediates, exemplified in the last section.
Finally, it is noteworthy that the encapsulation of the metal centre within the
protein matrix and the exclusion of water from the site can minimize the
dissipation of the electrophilic properties of Zn 2 +, and thereby strengthen its
catalytic power and contribute to the stabilization of the structure [32,33].
Catalytic Zn
Metalloenzymes where the active site metal is not directly involved in a redox
change are catalysts that employ electrostatic strain distortion (ESD) effects as
the primary means for lowering the activation energy of covalent steps in the
catalytic cycle [25,32]. In the catalytic sites of most zinc enzymes of known
structure, the Zn 2 + ion is invariably co-ordinated by three amino-acid side-
chains (with at least one His) and one water molecule [17]. Two of these ligands
are located in nearby regions along the protein chain, thus providing a local
preformed 'bidentate' site conformation as the binding locus for Zn 2 +. The
relative location of the third residue is much more variable, hence imparting the
52
Table 2 Zn chemistry in zinc proteases (see Figure 1 legend)
Zn co-ordination: Zn co-ordination
inner-sphere ligands Zn stabilization Activation Substrate! flexibility and
and geometry and electrophilic of the intermediate Zn 2+ co-ordination
(sequence distance activation by outeT- catalytic stabilization and numbers along the
between residues) sphere residues H 2 0 molecule activation catalytic cycle
-i
Thermolysin - Hisl42 (3) His146 _ Zn 2 + _ Zn 2 + Displacement of H 2 O :I:
(19) Glu 166 (residue - Glu143 - His231, protonated towards Glu143 upon
m
[42,43] tD
X) + H 2 O (enhanced (residue B) substrate binding 0
- Approximate by substrate - Tyr157, protonated - E=4, ES=5, EI=5, EP=4 5Gl
tetrahedron binding) - Glul43
Carboxy- - His69 (2) Glu 72 - H-bond network _ Zn 2 + - Zn 2 + for inter- - Glu72 co-ordination .--~
(J1 ()
peptidase A (residue X) (123) Aspl42-His 69-Zn 2 + - Glu270 mediates and products shifts from bidentate to :I:
W m
[22,30,44] Hisl69 + H 2O - Arg127, protonated monodentate upon
- Pentaco-ordination (residue B) substrate binding s:
Ui
- Glu72: bidentate - Ser197 carbonyl - E=5, ES=4, EI=5, EP=4 -i
co-ordination - Glu270 ~
0
Metzincins [27] - His218 (3) His222 - Possible electronic _ Zn 2 + _ Zn 2 + - Displacement of H 2O N
(fibroblast (5) His228 (residue X) interaction between a - Glu2l9 - Well-defined H 2O towardGlu219 upon
"Z
()
collagenase + H 2O conserved Met (eCH ,) (enhanced molecule possible substrate binding
numbering [46]) - Trigonal pyramid and plane faces of by substrate [48] (residue B) - When present, Tyr (Zn2+
- Additional Tyr ligand His2l8 and His 222 binding) - Tyr (Zn 2 + ligand) ligand) side-chain shifts
possible (47) - H-bond network possible, protonated from Zn2 + inner-sphere to
(reported in fibroblast [47] CO of scissile peptide
collagenase [45]) - Glu219 ~~~~g ~g?n substrate
Leu235CO-His2l8-Zn 2+
Leu226CO-His222-Zn 2+
Glu
'!,)=C
I
!
:' (~,
Ii l'I
~ ! "-/
Arg
HiS}B_H
"- /
9'I
Substrate
binding.
r~!-H
B-H····· .. · .... · .. 7.Q=~
Tyr \ I ... :
I rx
HZ
H20 Zn H
/N-H . / n_:
" .. H-N~N~l\'-""X {HiS
Glu IO=-C His'"
C -n-'" : ..
-....
AsP side-chain ~ ~~jJ A ..
\.
\ His H
or peptJde
carbonyl !/
! ,/ T
His
I(),H
I 6"Tyr
eH3
s'
elf
, 2
Tyr
eH2
Met'
E ES
(a) (b)
1
Glu
peptide
carbon~t ,;- "-
~
o\orl)'9
.... !
01
~
f"""", I
\! \'N-H
~~~~Ph": ]~t :=~~:O:d B-Hm:~~-,e-

p~
B-H ..
Proton
tranSfer. B-H ............... ~~;-
I/ : /1
I I I
Proton HZ / ! HZ
"':;-ZR: transfers n ' . / R_:
His'" -!-X His/' -,-X His'" i-X
His H His H His H
6"Tyr 6"Tyr 6"Tyr
EI EP EP
(c) (d) (e)
54
modulability required for substrate and chemical reaction specificities [23].

Therefore, the electrostatic field provided by the metal is tailored by the nature,
charge and orientation (which frequently deviates from regular tetrahedral
geometry) of inner- and outer-sphere residues and non-protein elements
(coenzymes and cocatalytic metals [34]) to (a) polarize substrates, (b) stabilize
negatively charged intermediates, and (c) modulate the pKa of a co-ordinated
ligand through ionization, polarization or displacement [25,35,36]. Indeed,
while the pKa of free H 20 is 15.7, this value is reduced to '" 10 in the
[Zn(OH)2))6f+ complex and to ",7 in the tetraco-ordinate [ZnNNO(OH2)d
model complex [37]. Such pKa perturbation opens the possibility for the
production of strong nucleophiles (e.g. OH-, R-Ol within enzyme sites at
physiological pH. For example, with three neutral His ligands, the co-ordinated
water molecule in carbonic anhydrase experiences strong polarization
(pKa ~ 7.0). Hence, carbonic anhydrase operates via the nucleophilic addition
of OH- to CO 2. With two His and one neutralizing Glu negative charge,
thermolysin polarizes H-OH for nucleophilic addition on a peptide carbonyl
(see below). With one His and two Cys thiolates to counterbalance the Zn2+
charge, the ESD contribution in alcohol dehydrogenase is primarily responsible
for the activation of the ground states of the substrate and product ternary
complexes via polarization of the inner-sphere co-ordinated species. In so
reducing the activation energy for covalent steps, the ESD effect frequently
renders subsequent steps in the catalytic cycle rate limiting (e.g. the proton
transfer in carbonic anhydrase, the coenzyme dissociation in alcohol dehydro-
Figure 1 General catalytic mechanism for thermolysin, carboxypeptidase A and Metzincins. At

each step, the interactions common to the three enzyme types are indicated by bold plain lines for
Zn2 + co-ordinate bonds and hashed lines for hydrogen bonds. Interactions occurring in the
mechanism of only one type of enzyme appear as dashed lines (see Table 2). X represents the third
His or Glu side-chain ligand of Zn2+, B-H stands for the protonated side-chain of an Arg
(carboxypeptidase A [44]), an His (thermolysin [42]), a Tyr (thermolysin [42]), or for a structurally
well-defined water molecule (in certain Metzincins [48]; see Table 2). In certain Metzincins, an
additional Tyr side-chain completes the co-ordination sphere to a pentaco-ordinate geometry
[27,47]. The peptide bond of the substrate is identified by thick bonds and a specific letter font. (a)
The catalytic site of the enzyme, E. The basicity and orientation of His ligands may be modulated
by an H-bond interaction with an Asp side-chain [22,30] or main-chain carbonyl [46], and/or by
electronic interactions between the imidazole ring 1t electrons and the E-methyl of a Met residue
[27]. The catalytic water molecule is stabilized, oriented and polarized by one or two [42] H-bond
interactions with the catalytic Glu side-chain. (b) Upon formation of the protease-peptide
substrate complex, ES, the catalytic water molecule is squeezed against the catalytic Glu, thereby
enhancing its polarization. (c) The tetrahedral intermediate, EI, resulting from nucleophilic attack
of H 2 0 on the activated electrophilic carbon centre of the substrate carbonyl is stabilized by
several interactions on both oxygen atoms and on the nitrogen atom. In carboxypeptidase A, the
main-chain carbonyl group of Ser 197 forms an additional H-bond to the intermediate hydroxyl
group [44]. (d) The concerted peptide bond breakage and proton shuttle to the leaving amino group
via the catalytic Glu residue lead to the formation of the enzyme--carboxylate product complex,
EP. (e) A second proton transfer to the leaving amino group is necessary to restore the ionized
form of the catalytic Glu side-chain [42]
55
genase) [25]. Therefore, the ESD effects are optimized to reach a compromise
between substrate activation, inner-sphere ligand exchange rates, and substrate/
product diffusion to and from the site. Noteworthy enough, such efficient
catalysis is well-suited for the activation of small substrates like CO 2 and
ethanol.
Non-catalytic Zn
Non-catalytic zinc sites are frequently organized as mononuclear or multi-
nuclear centres, in which the co-ordination polyhedron of Zn 2 + is a regular
tetrahedron excluded from the solvent and dominated by Cys thiolate ligands
that provide high stability to the complex. The thermodynamics of metal-ion
binding (see above) allows these relatively short peptidic sequences (usually
shorter than 60 residues) to fold into stable, structurally diverse domains, like
disulphide bonds do in many proteins [7,38]. Amongst these, are the famous Zn-
fingers found in the transcription factor IlIA (TFIIIA). These domains belong
to proteins responsible for a wide array of biological functions (enzymes,
transcription factors, hormone receptors, viral proteins). For example, they
promote (a) nucleic acid recognition (e.g. TFIIIA-type Zn-fingers, steroid-
thyroid receptors, Gal4 domain), (b) protein-protein interactions (e.g. the LIM
domains [39]), or (c) subunit oligomerization (e.g. aspartate transcarbamylase,
steroid-thyroid hormone receptor, bacteriophage T4 helix-destabilizing protein)
or domain-domain interactions (e.g. for the protein kinase C regulatory domain
[40]). In most cases, Zn 2 + mediates macromolecular interactions through the
stabilization of the interfacial protein surface. However, 3-dimensional struc-
tures are available for two particular complexes in which Zn 2 + cross-links
individual proteins: the growth hormone-prolactin receptor complex [11]; and
the insulin hexamer, the insulin form involved in the storage and processing of
the hormone [8,9,28], which, in addition, shows allosteric behaviour, as a result
of conformational equilibria between tetrahedrally and octahedrally zinc-co-
ordinated states [41].
AN EXAMPLE: ZINC PROTEASES

Owing to the importance of zinc proteases in a variety of biological processes,
including reproduction, digestion, hormone processing, embryonic develop-
ment, tissue differentiation and connective tissue maintenance, and of disorders,
including tetanus, botulism, snake-venom toxin-induced tissue necrosis, cancer
metastasis, rheumatoid arthritis and periodontal disease, zinc proteases provide
a good example for the purpose of illustrating the specific properties of Zn 2 + ion
in proteins. The 3-dimensional structures of several members of this family have
been solved [17], and the structures and catalytic mechanisms of thermolysin
[42,43] and carboxypeptidase A [30,44] have been thoroughly discussed, thereby
providing a framework for the understanding of related enzymes. These
mechanisms show convergent functional characteristics which are summarized
56
in Figure 1 and Table 2, along with the corresponding features of the

Metzincins, a subfamily of metalloproteases which includes the matrix metallo-
proteinases [27 and references therein]. The various characteristics and substrate
specificities of these enzymes reveal the adaptability of zinc chemistry in terms
of ligand nature and spacing along the protein sequence, co-ordination
geometry, and electronic properties (Table 2). For example, the polarization of
the scissile substrate carbonyl provided by the unbalanced Zn2 + charge of the
Metzincins (co-ordinated by three His, no Glu) appears strong enough to avoid
the participation of an additional protonated side-chain. However, in the
carboxypeptidase A system, co-ordination by two His and one Glu gives a site
which requires an additional positive electrostatic contribution by the proto-
nated side-chain of Arg 127 (Table 2). In peptide deformylase, the co-ordination
geometry of the catalytic zinc is compromised of two His and one Cys side-
chains, which imply another different adaptation of Zn 2 + chemistry within the
enzyme site for the catalysis of peptide bond hydrolysis [33].
Finally, the members of the Metzincin subfamily (Table 1) also take
advantage of the functional versatility of zinc during activation of the
proenzyme to the active enzyme. In the proenzyme, the Zn2 + ion, co-ordinated
in a Zn(N3S) tetrahedral ligand-field, is catalytically inhibited by the stable co-
ordination of the Cys contributed by the pro sequence. Upon conversion to the
active enzyme, this ligand is replaced by the catalytic water molecule [27,45].
Although the precise in-vivo activation mechanism has not yet been elucidated,
the cleavage and removal of the prosequence switches the zinc ion from a
structural co-ordination motif with four amino-acid ligands derived from the
protein, to a catalytic co-ordination motif usually comprised of three amino-
acid ligands and one catalytic water molecule.
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58
34. ValJee BL, Auld OS. New perspectives on zinc biochemistry: cocatalytic sites in multi-zinc
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35. Bertini I, Luchinat C, Rosi M, SgamelJotti A, Tarantelli F. pKa of zinc-bound water and
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Chern Res. 1988;21:333-40.
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45. Springman EB, Angleton EL, Birkedal-Hansen H, Van Wart HE. Multiple modes of
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59
5
Copper and zinc metallothioneins
V Albergoni and E Piccinni
Department of Biology, University of Padova, via U. Bassi 58/B,
35121 Padova, Italy
INTRODUCTION
Metallothioneins (MTs) are a family of ubiquitous and unusual proteins which
have been receiving extensive interest from chemists and biologists for the last
40 years. The first MTwas discovered in 1957 by Margoshes and Vallee [1] in
their search for a tissue component responsible for the natural accumulation of
cadmium (Cd) in equine renal cortex. MTwas so called because of its extremely
high metal and sulphur contents. Aside from Cd, this protein binds other
metals, especially zinc (Zn) and copper (Cu). Subsequently, researches of MTs
have greatly expanded, and their presence has been demonstrated in animals,
plants, fungi and cyanobacteria. MTs are characterized by the following
chemical properties: (a) low molecular weight; (b) high metal content; (c)
characteristic amino acid composition (high cysteine content, no aromatic
amino acids); (d) characteristic distribution of cysteinyl residues; (e) spectro-
scopic features characteristic of metal thiolates, with arrangement of metal ions
in clusters [2]. An appreciable number of MT sequences are now available.
Because of the variations in their primary structures, especially location of
cysteine residues and mode of synthesis, MTs are divided into three classes [2].
Class I comprise polypeptides closely related to the equine renal MT (location
of cysteine similar to that of horse kidney). This includes all mammalian MTs
including those from other vertebrates studied to date as well as a few from
some invertebrates, such as lobster, oyster, and mussel. Class II, comprise
polypeptides with location of cysteine only distantly related to that of horse
kidney. The 20 known sequences belonging to this class include protists, some
invertebrates (such as Drosophilia and sea urchin), cyanobacteria, yeasts and
some plants. Their lengths vary from 25-101 residues and lack homology, not
only among one another, but also with those of Class I. Class III are atypical
MTs that are not proteins, are not translationally synthesized metal-thiolate
polypeptides that contain y-glutamyl cysteinyl units. The class is found in plants
and some fungi.
Biosynthesis of MTs may be induced not only by many metal ions, including
the essential ones like Zn and Cu, as well as toxic ones such as Cd, Hg and Co,
61
but also by hormones, many cytotoxic and inflammatory agents, and a variety
of stress conditions [3].
TYPES OF MTS
Various kinds of isometallothioneins are common in animals, and may differ
only with regard to a few amino acids. The functional significance of mUltiple
forms of MTs still has to be clearly defined. Some of them are tissue specific and
may be related to differences in metal requirements during the life cycle. The
isoforms are encoded by different genes. As in other mammals, human MTs
(hMTs) occur in two electrophoretically distinct fractions, MT-I and MT-2. The
most complex polymorphism is found in liver, where the MT-I fraction contains
at least six subforms [4]; MT-2 is almost homogeneous. Thus, 12 isoforms have
been detected in humans until now - some of them tissue specific, such as the
recently discovered MT-3 in human brain (see later) and MT-4. Analysis of MT-
4 expression has been studied in mouse, in which it is expressed exclusively in
stratified squamous epithelia associated especially with oral mucosa, oesopha-
gus, upper stomach, footpads, tail and neonatal skin, In-situ hybridization
experiments indicate that MT-l and MT-4 are variously expressed during the
differentiation of these epithelia. Some data also indicate that MT-4 may play
some Zn-regulating role during such differentiation [5].
Mammalian MTs have a molecular weight of 6000-7000 Da, usually contain-
ing 60-63 amino acids (68 residues in mammalian brain subform MT-3 [6,7],
one-third of which are cysteines, frequently occurring in Cys-X-Cys and Cys-Cys
arrangements. These proteins show almost complete conservation of their
arrangement; the 20 cysteines are invariant, and Lys and Arg are also highly
conserved.
MTs isolated from organisms that have been experimentally exposed to
inducing'levels of heavy metals contain predominantly, but not exclusively, the
administered metal. For example, MT-2 isolated from the liver of Cd-treated
rats contains 5 Cd and 2 Zn [8].
STRUCTURES
The three-dimensional structure of MT was first studied on native Zn-Cd and
on the reconstituted form of Cd rat liver MT-2, by the application of a large
variety of spectroscopic methods [9]. Subsequently, structures for other mam-
mals, including man, were resolved. More recently, X-ray diffraction has shown
a crystal structure, in good agreement with the proposed dissolved state of the
protein. These methods confirm that the metal ions are tetrahedrally co-
ordinated to four cysteine thiolate ligands and partitioned into two distinct
metal clusters, B and A, binding 3 Cd (or 2 Zn+ 1 Cd) and 4 Cd ions,
respectively. The A-cluster is contained in the carboxyl-terminal ex-domain and
the B-cluster in the amino-terminal ~-domain. The folded protein is thus
organized into half molecules (domains) with limited contact, the connectin
62
COPPER AND ZINC METALLOTHIONEINS
being only in the Lys 30-Lys 31 segment. This characteristic, absence of an

interdomain gives independence to the folded domains that link metals with the
same stoichiometry as in the intact molecule; consequently, the flexibility and
plasticity that are two of the properties of MTs, may explain some fundamental
functions in the cellular environment, e.g. exchange and transfer of metals
[3,10,11].
In contrast to clusters involving divalent metal ions, there is little structural
information about Cu+ (or Ag+), which bind to MT as monovalent ions. The
stoichiometry is 12 atoms of Cu+ per mole of MT, and each domain forms
clusters containing 6 Cu+ ions. This suggests that binding is trigonal (rather
than tetrahedral, as in Cd2 +). Available data indicate that the N-terminal B-
cluster is arranged as a trigonal biprism [12]. The C-terminal domain is more
complex and its structure is not yet completely clarified, although recent data
indicate the trigonal co-ordination of all 12 Cu+ ions bound to the 20 cysteinyl
thiolates [13].
MT isoforms can exchange bound metal ions by direct transfer between
clusters, among isoforms, and among metallothioneins of different species [14].
Exchange is mediated by intermolecular ligands during transient association of
the MT molecules [3]. Although biological significance of molecular ion
exchange among MT isoforms is unclear, the high reactivity of the domains is
essential in understanding the roles of metal transfer and regulation played by
MT in the cellular and tissue environments. This view is elegantly supported by
some in-vitro studies on the relationship between thionein and zinc finger
transcription factors [15]. The effectiveness of this exchange in in-vivo systems
remains to be demonstrated.
BIOSYNTHESIS
Human MTs are encoded by a multi gene family (14 MT genes including non-
functional pseudo genes) located on chromosome 16 [16]. Seven are expressed
and identified in human liver [3]. As expected from the conservation of
mammalian MT protein structure, the coding DNA regions show good
homology. Functional MT genes show a tripartite structure, in which two
introns interrupt three exons at homologous positions at amino acid 9 and 31,
and all the genes contain the typical polyadenylation signal- AATAA - in the 3'
untranslated region [17].
Mammalian MT gene promoters are structurally complex. Induction by
metals is mediated by multiple cis-acting DNA sequences termed metal
response elements (MREs), representing binding sites for trans-acting factors,
proteins, regulating the level of transcription of MT genes in response to metal
concentrations.
Most studies on MT induction have focused on MT mRNA response and
identification of the corresponding cis-active elements. The human promoter
contains numerous sequences (MREs) upstream from the start transcription
site - TATAA - box: a G-C rich sequence (G-C box), two basal level enhancer
63
elements (BLEs) and metal responsive elements (MREs). These sequences are
found in either orientation and contain G-C rich sequences [18]. The MT-2 gene
promoter also contains a glucocorticoid-responsive element and a putative
interferon-responsive element [19,20], as well as regions that bind factors such
as activator protein-l and -2, (AP-I and AP-2) which activate the transcription
by inducing agents different from metals [21,22].
In mammals, the trans-acting factors, referred to as metal transcellular (or
transcription) factor (MTF), metal responsive (or regulatory) factor (MRF),
and metal binding factor (MBF) vary considerably; various molecular weights
have been reported but none has been fully characterized. The effects of different
metals on binding activity are variable, and it is unclear whether all metals that
induce MT synthesis also stimulate the same MTF. The data suggest that
multiple proteins are simultaneously present, capable of recognizing different
cis-acting elements (MREs) in the gene promoters in response to a specific metal
[17,23].
Data from several human cell lines indicate that the expression of iso-MT
genes is very complex. It is under separate control and may serve various
cellular purposes. In fact, the expression of iso-MT genes may vary in response
in particular tissues or cell lines, upon exposure to different MT-inducing agents
[24,25]. Only some examples will be reported here. Metals are the common
inducers of MT isoforms but, as noted above, a variety of agents has been
shown to induce MTs, and this has been demonstrated especially for the human
MT-2 (hMT) gene. This high inducibility probably reflects the greater complex-
ity of the promoter region of the hMT-2 gene with respect to that ofhMT-l. In
fact, only metals have been shown to induce hMT-l isoforms (with the
exception of the glucocorticoid-responsive hMT-l E gene).
Another factor has been demonstrated to regulate the different expression of
iso-MTs in tissue DNA methylation, involved in non-expression. This has been
demonstrated by various experimental approaches in MT-I or MT-2 non-
expressing cell lines. Direct evidence of MT regulation by methylated regions
has also been obtained by restriction experiments of genomic DNA from non-
expressing cell lines [25-28].
A recent model for the induction of MT transcription in mammals supports
the view that MT gene expression is regulated by Zn-mediated release of an
inhibitor that is bound to an MTF. The MTF is now ready for binding to MREs
and initiates transcription of MT which links other metals like Cd, Hg, Cu, etc.
These metals cannot activate MRFs (MTFs) directly but have great affinity for
Zn [29,30]. Further details on the molecular biology ofMTs have been reviewed
in recent papers [23,31].
A new member ofthe iso-MT family, MT-3, was identified in human brain by
Uchida and collaborators [6] while studying Alzheimer's disease (AD). Because
it inhibits brain cell growth and neurotrophic activity, this member was called
growth inhibitory factor (GIF). The authors demonstrated that the abundance
ofneurofibrillar tangles is correlated with the level of this protein, that GIF is an
MT (with two unusual inserts) located in astrocytes in the grey matter, and that
64
it variously decreases in the AD brain. The purified MT contains 3 Zn and 4 Cu

per mole of protein [6]. However, it is possible that the protein is not fully
saturated, since previous experience has shown that conventionally purified
Cu-, Zn-MTs do not always reflect the true potential saturated state of MT-3
[11]. This protein has 68 amino acids, 38 of which are identical to human MT
isoforms, giving about 70% sequence similarity with mammalian MTs, includ-
ing the 20 cysteine residues that are completely conserved. One of the major
distinctions with respect to other mammal isoforms is the presence of one the
onine. in position 5 and six glutamic-rich residues inserted in position 55 in the
C-terrninal domain. The presence of eight glutamic residues makes the apo-MT
acidic. MT-3 isoforms with similar characteristics have been obtained from
other mammals [7,32]. Although the six-residue insert in the ex-domain is not
fully conserved, its composition suggests a helical structure. Analysis of
differences between MT-3 sequences and MT-isoforms suggests a substantially
more tightly folded structure of GIF. In contrast to MTs which bind 7 divalent
ions Zn and/or Cd and up to 14 (monovalent) Cu+ ions, native MT-3 contains
Cu and Zn in species-specific ratios [32]. Therefore, metal binding may be
functionally important due to its unique biological inhibiting activity, which still
remains to be clarified. The coding gene for MT-3, similar to the other MT-l
and MT-2 isoforms, is clustered on the same chromosome 16 in the human (8 in
the mouse). The 5' and 3' untranslated regions and promoters show little identity
within the three isoforms [7].
CONTENT, LOCATION AND INDUCTION

Basal MT levels are found in most adult mammalian tissues. Data on species
variations reveal that human, dog, cat, pig and goat have the highest levels (700-
400 ~g/g liver). In other animals (e.g. monkey, sheep), the values are lower
(about 200 ~g/g liver), the lowest (2-10 ~g/g) being found in mouse, rat,
hamster, rabbit and guinea-pig. Cu and Zn contents show similar trends [33].
The levels may vary with age and type of tissue and depend on nutritional and
physiological factors, as well as pathological conditions. Data on MT mRNA
levels indicate that tissue-specific MT synthesis occurs primarily in the organs of
absorption and excretion, suggesting that MT plays a role in the physiological
control of these processes [34]. MT also occurs in blood (plasma, cells and
platelets) [35,36]. At the cellular level, MT contents occur mainly in cytoplasm
and, to a lesser extent, in the particulate fraction, mainly in lysosomes and
nuclei [37,38].
The kind of metals bound reflects natural exposure and the expression of
various MT isoforms. For instance, in human liver, MT is mainly present as Zn-
MT, also linking Cu, and is one of the major Zn proteins, but in Cu-Ioaded
animals, MT is the major Cu-binding protein, and Zn is secondary [39].
CU,Zn native MT is present mainly in fetal and neonatal liver in man and
other mammals [40] and decreases to basal levels during growth [41]. It has been
demonstrated that the early expression of MT initiates in the endodermal cells
65
of mouse embryo [42]. Changes in MT levels and cell localization in mammalian

liver during development and its presence in some undifferentiated tumours
suggest that MT plays a role in cell differentiation and maturation [43]. It is also
assumed that MT expression in the fetus may indicate a shift from proliferation
to differentiation [44]. Increased MT and metal accumulation in fetal and
neonatal life may be explained by high requirements for essential elements for
metabolic processes. It is interesting to note here that MT is present in human
fetal liver, even at the nuclear level, whereas in adult humans it is not found.
Flow cytometric analysis indicates MT translocation from cytoplasm into nuclei
in regenerating liver after partial hepatectomy. In this tissue, MT induction
occurs and protein levels are about 80 times higher than in intact tissues [45].
Nuclear location has been related to Zn requirements and interactions with
nuclear constituents during the cell cycle [41].
Metals, glucocorticoid hormones and cytokines are very effective in MT
induction, and are also used to evaluate local variations in MT content.
In comparison with Zn, Cu is a poor inductor of MT synthesis, but it may
replace Zn in MT, as also shown in vivo [46]. Released Zn may be active in
inducing new MT synthesis. Other data confirm independent MT induction by
Cu, as verified in injected rats in which MT mRNA levels increase prior to the
increase in hepatic Zn uptake [47]. Data on MT mRNA in liver confirm that Cu
is less effective in increasing mRNA. However, the induction is not linear, and
at high doses the effects of Zn and Cu are similar [48-50]. It has recently been
observed that a Cu-deficient diet severely depresses Cu concentrations in liver,
kidney and heart, and markedly increases MT-l mRNA only in liver [51]. Data
deal with the relationship between the physiological condition of Zn and Cu
content in the diet and MT induction. When these trace elements are present in
sufficient amounts, accumulation and redistribution of metals occurs in intes-
tine and liver, with increased amounts of MT. The conformational differences
and binding characteristics of MT sites may be involved in the metabolism or
homeostasis of Cu and Zn [52].
The effects on MT levels of Zn, stress and endotoxin treatments in rat have
been analysed together with the interactions between these inducers. Synergistic
effects in animals but not in primary cultures of hepatocytes suggest that the
observed synergism between Zn and other inducers in MT synthesis in the liver
may be a consequence of increased Zn levels in the body [53]. In a variety of
chemical or physical stress conditions, in the acute phase response, the
expression of MT genes (together with other specific genes) in the liver is
probably mediated by interleukin-l secreted by macrophages or other lympho-
kines [54].
Besides steroid hormones, various commonly used non-steroidal anti-inflam-
matory drugs cause an increase in MT content in the liver, either modifying the
tissue distribution of Zn or acting as radical oxygen scavengers [55].
The amounts of MT-3 mRNA in mouse brain are unaffected by treatment with
Zn, Cd, dexamethasone (DEX) or lipopolysaccaride (LPS). These components
induce MT-l in most organs but only the latter two, and to a lesser extent Cd, are
66
effective inducers in brain. The data are confirmed by expression results on

murine astrocyte cell cultures, demonstrating that also in this condition MT-3 is
unresponsive to induction with respect to MT-l and MT-2 (these isoforms
respond to Cu and Cd and especially to DEX). The data suggest that the blood-
brain barrier is not the only limiting feature for induction ofMT-3, as previously
suggested by Palmiter et ai. [7). DEX and corticosterone stimulate MT synthesis
in rat brain, but their roles in the regulation of protein levels differ between tissues
and within specific areas. There is evidence for a complex role of glucocorticoids,
Zn and Cu in controlling MT accumulation [56). Analysis of MT-I and MT-2 is
more complicated due to their unequal induction in various animals and, since
MT-l and MT-2 are less expressed in brain than in hepatic tissue, other tissue-
specific factors are involved in MT gene expression [7,57-59).
Induction of MTs have been obtained in human skin by UVB irradiation,
indicating their physiological protective role in human skin cells [60], a role
confirmed by data from various strains of UVB-irradiated human cell cultures
and showing that the strains in which MTs are induced are less sensitive to
irradiation. According to Kobayashi et aI., the protective role is similar to that of
GSH [61]. Since UV irradiation of skin induces synthesis of superoxide dismutase
(SOD), this MT induction is functionally additional to SOD as a Cu-dependent
antioxidant [62,63]. A factor secreted by irradiated cells has also been shown to
induce MT mRNA in non-irradiated cultured human fibroblasts [64].
It has recently been reported that MT synthesis may also be induced in mouse
liver by exposure to static magnetic fields. This effect is cumulative to those
obtained by treatment with carbon tetrachloride [65).
DEGRADATION AND TURNOVER

MT degradation is poorly defined since in-vitro and in-vivo results are not
completely comparable, partly due to in vivo measurement difficulties. Protein
degradation may occur in both cytosol and lysosomes, whereas the nuclear
location preserves the protein, reducing its availability to cytosolic or lysosomal
degradation [66). Experiments in vitro and on apo-MT indicate that the greatest
degradation rate occurs in lysosomes, being about 400 times lower in cytoplasm
at optimal pH values of 5.5 and 7.2, respectively. The proteases involved are
cathepsin Band/or L, which shows the greatest activity [67). Zn-, Cu- or Cd-
MTs are more resistant to in-vivo hydrolysis than apoprotein [68,69). MT
degradation therefore seems to be regulated by the amount or by the different
bound metals. Metal displacement depends on pH value, being related to
binding affinities. Differences in the degradation of MT-l and MT-2 have been
demonstrated: MT-2 is more resistant [67], indicating the importance of protein
degradation in regulation in MT concentration [70]. Degradation of the ct-
domain in Zn-Mt-l and of the ~-domain in MT-2 may also occur independently
[71].
The particulate granular form of Cu-MT, polymerized in insoluble aggregates
found in liver lysosomes, have a degradation rate lower than the cytosolic form
67
[72,73]. In in-vivo experiments some divergence exists, leading to the conclusion

that some additional factor, independent of structure stability, influences MT
degradation [67].
ROLE
It is very difficult to define the specific function of MTs, due to heterogeneity of
their involvement in various physiological and pathological conditions. This has
been provokingly indicated by Karin [74] in the title of the paper "Metallothio-
neins: proteins in search of function".
The presence and conservative characteristics of Class I MTs in various phyla
may mean that this protein plays an essential role, or roles, in organisms. The
problem is, with reference to the initial stimulus, to distinguish between primary
and secondary involvement. Many functions have been proposed or presumed
until now, but only the effects of the presence of MTs and correlated induction
have been well documented. Many data exist on the role of MTs in metal
metabolism and metal regulation, although it is not always easy to distinguish
between these and the other effects or roles of this protein.
Regulation of MT biosynthesis controlled by metals has been considered as a
biological device to control the concentration of free metal ions, both essential
and non-essential, by their chelation [75]. The capacity to sequester metals may
be exploited to store them, particularly during the development and growth of
the organism.
Certainly, many essential metals are bound according to the affinity constant,
and this bond therefore plays a functional role for some metals. MTs may
therefore also be considered as metal buffers involved in steady-state kinetic
maintenance of intracellular Cu-Zn levels [76]. In other conditions, when
competition between essential and non-essential metals on the same MT site
occurs, owing to their different affinities and/or concentrations, the bond must
be considered accidental and with no specific physiological role. In this case, the
flexibility of the molecule and the exchange of metals are essential.
The putative role of MT as metal transporter or metal donor has been
debated. In-vitro experiments indicate that metal is released in oxidizing
conditions. Metal transfer from MT to various enzymes has been accomplished
in in-vitro and well-defined conditions. In any case, MT is involved both in
metal reserves, as indicated by its levels in the life cycle, and as a metal buffer, as
previously mentioned. There is no in-vivo evidence for direct transfer of copper
to apoenzymes.
The characteristics of the two binding domains of MT and their importance
in acting as metal donors and metal acceptors have been investigated, and MT
has thus been hypothesized to rescue metal-intoxicated metalloproteins or
metalloenzymes [77].
Recent data have demonstrated the existence of a natural oxidative chemical
process able to mobilize zinc from MT, in which MT interacts with glutathione
disulphide, with concomitant release of the metal. In-vivo redox control of Zn
68
distribution has been proposed, with MT as Zn donor [78,79]. The existence of

intracellular Cu(I)MT-GSH and Cu(l)-GSH complexes in mammalian cell
lines has been reported. Cu(I)-GSH may derive from the complex with MT in
Cu-deficient conditions, and has been proposed as an intracellular donor of Cu
[80,81]. Other data on binding characteristics support the presence of a GSH-
Cd,Zn-MT binding site [82].
The role of MT as Zn donor in DNA replication occurring in tissue
regeneration is supported by data from regenerating liver in which the presence
of previously Cd-induced MT causes inhibition of the first regeneration peak
[83].
Data regarding a subclass of glutamatergic neurons mainly present in the
telencephalon, which contains Zn at presynaptic terminals, are particularly
interesting due to the possible functional role of MTs in neuronal signals.
'Vesicular' Zn pools are reviewed as endogenous modulators of ligand- and
voltage-gated ion channels, in which Zn is released by a calcium-dependent
mechanism. MT isoforms that have also been quantitatively determined are
thought to operate the mechanisms to donate, distribute and sequester zinc at
presynaptic terminals [84]. Because a correspondence has been found between
MT III-mRNA and neurons which store Zn in their terminal vesicles, Masters
et al. suggest that MT-3 too plays a role in the neuromodulation of Zn [85].
In intestinal absorption, a direct relationship has been found between Zn
absorption and Zn bound to MT, possibly indicating a functional relationship
between MT content and transfer of Zn to plasma. Following conflicting data
and new knowledge [86], these initial conclusions have been revised and a model
proposed, in which cysteine-rich intestinal protein (CRIP) and MT may
interact. CRIP facilitates transport across enterocytes and passage through the
basolateral membrane [87] and MT may inhibit Zn absorption by binding Zn in
competition with CRIP.
As regards the role of MT in copper absorption, no direct correlation
between Cu in the diet and mucosal MT concentration is evident [88]. During
oral Zn treatment, MT contents in intestine increase about 5-fold after about 2
weeks, returning to normal at about 5 weeks [89]. Other data on rats orally
treated with Zn for 4 weeks show an MT increase of about double in liver and
intestine, where Cu contents are unchanged. Contemporary oral and parenteral
Zn and Cu administration results in a decrease in Cu content in comparison
with Cu treatment only, whereas the MT level does not seem to depend on Cu
treatment [90,91]. These data suggest that some other control mechanisms are at
work in rat, probably not involving MT alone, to reduce the amount of Cu in the
body. In other studies, a significant reduction in Cu status has been described in
animals treated with high levels of Zn, but data are conflicting [92,93].
Interesting results have recently been reported on Cu transport from mucosal
to serosal intestinal sides and then to liver. After direct Cu(II) administration in
the jejenum, Cu and MT levels are clearly higher in portal blood than in the
vena cava, indicating that MT may transport cuprous copper [94]. In the various
experimental approaches confirming the effect of Zn on Cu status, the
69
mechanisms may be different, involving not only possible interaction in the

entering metal but also in Cu excretion. The obligatory role of MT in Cu uptake
has not been documented. This is improbable since other cytosolic proteins are
involved in initial Cu binding [95].
MT may act as an excretory molecule in the control of metal homeostasis,
since Cu-MT is partly excreted in biliary ducts and secreted in plasma. In pigs,
there is a direct correlation between liver and bile Cu concentrations [48]; in
sheep and other animals, like rat, there is an inverse correlation, and only a
small proportion of hepatic MT is directly secreted into bile. In any case, MT
may be excreted intact with its metals [39,96]. MT is also involved in exocrine
pancreas secretion, as clearly found in mouse by means of MT immunocyto-
chemical location in acinar cells and pancreatic ducts but not in islets.
Immunolocation also indicates that MT is present in cytoplasm but not in
secretory vesicles. Increased MT levels have been reported in pancreatic juice
stimulated by pilocarpine. In transgenic mice, which overexpress MT-I, and in
Zn-induced MT in both normal and transgenic animals, data indicate MT
location only in acinar cells and pancreatic ducts. In stimulated secretion of
pancreatic juice, MT contents are increased, especially in Zn-induced MT, in
comparison with those in MT-overexpression in transgenic animals. These data
suggest the physiological role of pancreas in Zn homeostasis [97]. No quantita-
tive data exist on the loss of MT through intestinal mucosal cells, including cell
loss by continuous desquamation, or on MT digestion (if it occurs), with an
estimate of subsequent metal reabsorption.
Plasma MT is filtered through the glomerular kidney membrane and partially
or totally reabsorbed by pinocytosis in the proximal tubule, according to the
aspecific mechanisms which operate in both these functional steps. The only
possible specific physiological mechanism of MT excretion may be tubular
secretion, which, however, has never been reported.
Data on metal absorption, binding equilibria and excretion indicate that MT
is certainly involved in essential metal metabolism, and a general model
describing the MT role in Cu and Zn cellular metabolism has been illustrated
[48,76]. Data on the biological functions, induction and degradation of MT has
recently been extensively reviewed by Cheri an and Chan [98].
A comparative analysis of the adaptive response of animals to essential and
non-essential metals illustrates the various mechanisms and compartments for
regulation and accumulation, and the putative role of MT has been discussed
[99].
The different cell distribution of iso-MTs in brain tissue suggests specific
functions for these proteins in various brain regions [58]. Results indicate that
the expression of MT-3 is regulated in a fashion different from that of MT-I and
MT-2 [59], so that the previously quoted inhibition model [29] for MT gene
expression should be revised for MT-3. As previously reported, the first function
for MT-3 is neurone growth inhibitory activity [6], and, because mRNA MT-3 is
down-regulated in patients suffering from Alzheimer's disease, this protein is
implicated in the aetiology of this complex neurological disorder [100]. How-
70
ever, the important role typical of the other MTs, in metal homeostasis of the
brain and in neuroprotection, may also be assigned to MT-3. In addition, the
position of the astrocytes, between capillary endothelium and neurones, endows
MT-3 with properties of neuronal protection against chemical injury.
As regards the cytoprotective role of MT, in-vitro experiments on rat
hepatocytes indicate that overexpression of MT, obtained by Zn or Cu
treatment, protects cells from cytotoxic effects caused by alkylating agents. As
for the mechanism involved, a role for MTs as antioxidants and electrophilic
species scavengers has been suggested [101]. Experiments on embryonic
fibroblast cells from transgenic mice deprived of MT expression due to
disruption of MT-l and MT-2 genes show increased sensitivity to cis-platin
and many other chemical compounds used as anticancer drugs [102,103]. It has
been demonstrated that the interactions between MT and nitric oxide produced
by inflammatory states protect against DNA damage and cytotoxic effects, as
occurs in MT overexpression [104]. In oxidative stress, MT can protect DNA
from damage caused by reactive oxygen radicals generated by copper but not
from those generated by iron. This protection seems to be due to chelation of
Cu, thus preventing its participation in redox reactions [105]. The cytokines
released during oxidative stress may at least partially mediate MT induction. In-
vitro experiments regarding the possible role for MT in protecting against DNA
damage by degradation of hydroxyl radicals have been performed using iron(II)
in the reaction mixture. The data clearly support the scavenging effect of MT on
hydroxyl radicals [106]. Other in-vivo experiments indicate that Cd-resistant
clones enriched in MT are significantly more resistant than non-enriched ones to
oxidative stress extracellularly generated by H 2 0 2 or H 2 0 2 and O 2- produced
by the xanthine oxidase-acetaldehyde system. No changes in catalase and
glutathione peroxidase and a decrease in SOD contents in clones have also been
reported [107]. Further data indicate that yeast and mammalian MTs have Cu-
dependent antioxidant activity, which substitutes that of Cu-Zn SOD. Data
obtained in vitro have been confirmed on monkey phenotypes associated with
SODI deletion strains [63].
Cytokines are released during oxidative stress and they may, at least partly,
mediate MT induction, as already mentioned. The induction of MT synthesis by
chemical stress is tissue specific, and liver is particularly responsive. The increase
in MT content is associated with various types of inflammatory or stress stimuli,
as well as in damaged tissues, so that damage by free radicals is prevented. The
anti-inflammatory effects of circulating or injected MT have also been reported
[54].
All these data on MT increases and effects and many evaluations, including
the broad inducibility of MT and the presence of various gene regulatory
elements, suggest that MT may be considered as a stress protein.
71
METALLOTHIONEIN AND THE IMMUNE SYSTEM

In-vitro experiments confirm that high MT induction rates by Zn occur in
isolated monocytes and in B- and T-lymphocytes. Zn and DEX produce similar
effects, with induction faster than that of Cd. Monocytes and B- and T-
lymphocytes vary in MT accumulation according to this decreasing order, in
which the increased values are proportional to background MT levels [108].
As MT is present in blood as a result ofthe normal process of cellular secretion,
mainly by liver [109], the correlation between plasma MTand the immune system
has been studied. In-vitro experiments clearly indicate that both extracellular
Zn,Cd-MTor extracellular apothionein stimulate lymphocyte proliferation, and
that this effect is synergistic with polyclonal activators of proliferation. The
mechanism ofthis response probably involves the interaction of MT thiols having
no metal bound to the lymphocyte membrane, which generate a calcium-ion flux
responsible for subsequent DNA synthesis and cell division [110]. Extracellular
MT bound to the plasma membranes of T- and B-lymphocytes acts synergisti-
cally with specific mitogens, inducing their proliferation, whereas, if only MT is
present, proliferation occurs in B-lymphocytes, enhancing their capacity to
differentiate into plasma cells [111]. Other researchers have also examined the
effects of extracellular MT on humoral responsiveness. In-vitro results indicate
that MT alone is capable of inducing a slight increase in IgM secretion, whereas,
in in-vivo experiments, LPS-induced IgM secretion is suppressed by Zn,Cd-MT,
thus indicating the presence of specific interactions between MT and some
components of the immunoresponse [112]. Although MT has no effect on the
phagocytic capacity of macrophages, at the highest doses it significantly
stimulates the production of oxygen radicals, thus augmenting the death rate of
phagocytozed micro-organisms. MT also significantly decreases the antigen-
primed T-cell proliferation stimulated by macrophages [113]. All these data
favour a possible physiological role of MT mediation in immunomodulation.
CONCLUSIONS
The role of MT in metal detoxification is undoubted, due to the capacity of
sulphydryl groups for metal trapping. Their importance is confirmed by the
relationship between metal tolerance and MT content, but it is unlikely that
protection against heavy metals is the primary function of MT, which probably
plays an additional rather than a primary role [35] and is aspecific. Further-
more, the basic level of MT expression is relatively high, and metals probably do
not exert sufficient selection pressure to explain the existence of a special
detoxifying system [74]. Conversely, data on prokaryotic metal resistance
suggest that life began in an environment polluted by products of volcanic
activity and other geological sources, so that heavy-metal regulation and
resistance was an early necessity. It is interesting to stress that, in bacteria,
membrane ATPases are active for heavy-metal homeostatic control. Chelating
molecules are also codified in bacteria, although they have not yet been well
72
characterized. Both MT and heavy-metal transporter ATPases are expressed in

cyanobacteria. Lastly, various MT isoforms are present in most eukaryotic cells,
but it is dubious whether heavy-metal transporter ATPases, similar to those of
bacteria, are expressed. In fact, two defective genes for Cu-transport ATPases,
one in Wilson's disease and the other in Menkes' disease, have been described
[114], indicating the putative presence of two heavy-metal transporters in
eukaryotes. Certainly, on the basis of genetic potentiality, in the course of their
evolution, organisms were able to improve and specialize different adaptive
strategies according to environments and specific conditions, so that MT
acquired a central role in eukaryotic organisms.
We may now summarize, and conclude that all the literature data confirm a
significant role for MT in metal homeostasis, both for binding characteristics
and induction mechanisms. Binding and regulation involve first Zn and then
Cu. Essential metals, Cu and particularly Zn play multiple roles in biological
functions and, consequently, MT too appears to be a multifunctional molecule
involved in various and only apparently unrelated responses. The wide range of
inducers confirms that MT may be involved and utilized in various tissues in
both normal and abnormal conditions, to maintain or restore the basic levels of
functional parameters in the body, or to complement other components. The
molecular characteristics of MT, particularly the arrangements of the two
distinct domains, ex and p, with peculiar metal specificity, confer multiple wide-
range biological roles on this family of peculiar proteins. Correlations between
minute structural differences and the predetermination of functional character-
istics are still unresolved and poorly understood [11]. Certainly, the existence of
a protein with the characteristics of MT, i.e. various isoforms and different
locations, transfer between different compartments, exchange of essential and
non-essential metals, and multiple agents of induction, has endowed organisms
with an efficient molecule showing unexpected and surprising properties.
ACKNOWLEDGEMENT
We thank Dr Paola Irato for her assistance.
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78
6
Zinc in the regulation and therapy of
inflammatory diseases and gastrointestinal
ulceration
KD Rainsford and B Zeitlin
Division of Biomedical Sciences, Sheffield Hallam University,
Sheffield, S1 1WB, UK
INTRODUCTION
Zinc is a key trace metal ion that is important in regulating a wide variety of
metabolic, hormonal, immunological, neuronal and epithelial cell functions [1-
4] (Chapters 2, 4 and 5). Over 300 enzymic reactions are known to depend on
the presence of zinc [3]. These roles of zinc may be considered to have
importance for (a) structural components of metalloproteins (e.g. in thymulin,
gene-regulatory proteins, steroid receptors, (b) catalytic activity of enzymes (e.g.
in various oxido-reductases, hydrolases, ligases, lyases) and (c) co-active
functions (e.g. with Cu in superoxide dismutase, or phospholipase C [3].
Zinc and copper are considered to act in opposite ways, although paradoxi-
cally in some conditions they may have pharmacologically apparent similar end
effects [5] (Chapters 2 and 4). Such is the case for the anti-inflammatory and
antigastric-ulcer effects of these drugs [5]. Superficially one might suspect
similar mechanisms of action of these two metal ions. However, with a few
exceptions, they do have synchronous biochemical or pharmacological roles
(e.g. in both having co-activity in Cu/Zn-superoxide dismutase (SOD) or in the
actions of SOD mimetics).
An important chemical property that differentiates the actions of zinc from
those of copper is their redox reactions (Chapter 4). Thus, the electron acceptor
property of zinc is such that it has high affinity for electron-donor molecules,
such as thiolates or amines, depending on their oxidation states. It therefore
readily forms complexes with amino, carbohydrate or thiol groups of amino
acids, peptides/proteins or their glycoproteins [5]. Copper is an electron donor
(depending on its oxidation state) and, while also having affinity for thiolates
and carboxylates, is generally more abundant in different biomolecules than
zinc. The mechanisms of the anti-inflammatory and anti-ulcer actions of zinc
would, therefore, be expected to differ to a large degree from those of copper
compounds based on chemical and biochemical actions of these metal ions.
79
In this chapter, the molecular, cellular and physiopathological actions of zinc

will be examined as they are relevant to the pathogenesis and treatment in
inflammatory conditions and gastrointestinal diseases. Some actions of zinc will
also be contrasted with those of copper in these states. Several books and
reviews on some of the roles of zinc and copper ions in these states have
appeared recently [5-7]. Therefore, it is not proposed to review the more general
actions of this metal at length but to concentrate on bringing together the recent
studies on the actions of zinc in inflammatory and gastrointestinal conditions,
particularly as they may be important for understanding the pharmacological
actions of novel zinc compounds or those agents likely to affect the actions of
zmc.
RATIONALE FOR ZINC THERAPY

Zinc deficiency
The basis for employing zinc compounds in the therapy of chronic inflamma-
tory diseases has rested in part on the suggestion that there is a deficiency in zinc
in the circulation, organs or inflamed tissues of rheumatoid arthritis patients [8].
Thus, it is supposed that a generalized zinc deficiency in chronic inflammatory
diseases would ipso facto cause a deficiency in certain key zinc-containing
enzymes or other biologically effective molecules that are important in the
regulation of immune or other cellular functions important in manifesting
inflammatory conditions. The evidence for these propositions requires close
scrutiny so that the critical changes induced in the different inflammatory states
can be identified.
Rheumatoid arthritis
Simkin [8] in 1981 reviewed the published reports in which serum zinc
concentrations had been determined in patients with rheumatoid arthritis and
compared with those in control subjects. There appeared to be considerable
geographical variability in the differences between these groups. Thus the
differences in zinc levels between patients with rheumatoid arthritis and controls
appeared greatest in the data derived from studies in India, New Zealand and
two locations in southern USA, whereas they were less so in data from Glasgow
and Omaha, and not significantly different in groups from Seattle, Rochester
and Parma [8]. The difficulty in comparing data from the various locations is
compounded by the fact that not all studies were case-matched and variations
would be expected according to a whole range of factors, patient-related,
methodological, and possible effects of therapeutic agents. Furthermore, the
techniques especially instrumentation, available then were probably less sensi-
tive and specific than those available today.
In a study reported by Frigo et al. [9] plasma concentrations of zinc
determined by atomic absorption spectroscopy were found to be reduced in
80
ZINC IN INFLAMMATION
patients in Verona with rheumatoid arthritis but not osteoarthritis compared

with controls. Moreover, the plasma zinc status was found to be correlated with
duration of illness, number of swollen joints, ESR and cx.rglobulins [9]. Urinary
elimination of zinc over a 24 hr period was not shown to be reduced in patients
with rheumatoid arthritis. These observations have been confirmed in later
studies [10].
Possible differences between the earlier studies, many of which were from
assays of serum samples compared with those observations in plasma samples,
could be related to the fact that serum samples are often obtained under variable
conditions of collection and the red blood cells can serve as a source of zinc
sequestered during formation of serum. Furthermore, plasma zinc levels
correlate with albumin which is the principal ligand for zinc in the circulation.
Since plasma albumin is often reduced in rheumatoid arthritis it is important to
establish if the reduction in plasma zinc. In controlled analysis of zinc profiles in
blood components in patients with rheumatoid compared with osteoarthritis,
Dore-Duffy and co-workers [10] showed that the statistically significant reduc-
tion in both plasma and serum zinc concentrations in the former but not the
latter patients was also observed in the fraction of albumin binding zinc (J.1g Zn
albuminig albumin) as well as in the plasma protein fraction. This suggests that
zinc affinity for albumin is somehow reduced in rheumatoid arthritis. No such
changes were reflected in the patients with osteoarthritis. Furthermore, no
changes were observed in the zinc concentrations in red blood cells or in white
blood cells [10]. The study by Dore-Duffy et al. [10] is notable for the fact that
the patients were asked to refrain from drug intake for 12 h prior to sample
collection and these samples were collected at 8-10 am to minimize diurnal
variation. The zinc was analysed by atomic absorption spectroscopy. Using x-
ray emission nuclear microprobe analysis, Svenson and co-workers [11] were, in
contrast to Dore-Duffy et al. [10], able to show marked reductions in zinc
concentrations in the granulocytes, platelets and erythrocytes of patients with
rheumatoid arthritis, seronegative spondylo-arthropathies and scleroderma.
Thus, the more sensitive and specific nuclear microprobe technique probably
accounts for the differences observed between the study by Svenson et al. [11]
compared with that by Dore-Duffy et al. [10]. The former authors also
confirmed the reduction in plasma zinc concentrations in patients with
rheumatoid arthritis, again using the more sensitive methodology of X-ray
fluorescence spectrophotometry. Negative correlations of plasma zinc concen-
trations with disease activity (ESR, serum orosmo mucoid and other biochem-
ical parameters) were again observed [11]; a feature which has been observed by
others in serum samples in rheumatoid arthritis patients assayed by atomic
absorption spectroscopy [12] as well as in plasma samples from patients with
psoriatic arthritis analysed by this technique [9].
In contrast to the observed reduction in zinc in plasma and blood leucocytes,
there is an apparent increase in zinc concentration in the synovial tissues of
patients with rheumatoid arthritis [8]. Explanations for this effect are not known
[8] but it could be that the increased cellularity from synovial proliferation
81
(pannus synovitis) produced as a consequence of the disease could lead to a

relative increase in zinc.
In an animal model of arthritic disease in rats induced by the injection of
heat-killed/delipidated Mycobacterium tuberculosis into the tail base (adjuvant
arthritis), it has been found that plasma zinc is decreased to a greater extent than
the reduction in body weight accompanied by an increase in hepatic metal-
lothionein and plasma copper [13]. Increased plasma copper has also been
noted in patients with rheumatoid arthritis [12]. The increase in hepatic
metallothionein has been observed in patients with rheumatoid arthritis [12]
but the correlation with hepatic metallothionein is less clear than the correlation
observed with plasma zinc [13]. Since hepatic and possibly intestinal metal-
lothionein synthesis is regulated by both zinc and copper (see Albergoni and
Piccinni, Chapter 5) [3] and transport of these metal ions is regulated by
metallothioneins, it is possible that alterations in zinc in the plasma and
circulating cells could be affected by effects of the inflammatory disease on
metallothionein synthesis. Thus, the observed increase in hepatic metallothio-
nein in adjuvant arthritic rats could be due to a compensatory increase in the
synthesis of this protein from zinc deficiency or actions of inflammatory
mediators (e.g. proinflammatory cytokines) affecting either zinc status or
metallothionein production [14].
Naveh and co-workers [15] recently reported studies in which they compared
the absorption of zinc (50 mg elemental zinc) in rheumatoid arthritis patients
with high and low disease activity with control subjects. They showed that both
plasma and urinary zinc levels were significantly lower in the two patient groups
compared with controls and concluded that this was evidence for malabsorption
of zinc in rheumatoid arthritis.
Overall, therefore, it would appear that the decline in plasma zinc concentra-
tions in rheumatic and possibly also psoriatic arthritis is probably related to the
combined effects of reduced intestinal transport of the ion and a reduction in
both the mass of albumin and its capacity to bind zinc ions. This reduced
circulating zinc in bound and free forms probably leads to the reduction in
leucocyte zinc concentrations that may have consequences for the control of
inflammatory and immunological functions by those cells. The elevated synovial
tissue concentrations of zinc may reflect the cellular proliferation and possibly
increased turnover of this metal ion as a consequence of degenerative changes
with consequent disposition of the metal in synovial fluids.
The main pharmacological actions of zinc may, therefore, reside at the level
of the leucocytes given the reduction in zinc in these cells. Additionally,
supranormal levels of zinc delivered to inflamed synovia might have specific
actions over and above the already higher levels in this tissue. These issues
remain central to understanding the sites and specificity of potential zinc-
containing therapies.
82
Osteoporosis
Zinc deficiency has been shown to reduce osteoblastic activity, collagen and
proteoglycan synthesis, as well as alkaline phosphatase activity in rats [16,17].
However, oral administration of high doses of zinc salts has been found to
stimulate bone resorptive activity in rats [18]. This suggests there may be
opposing effects of zinc on bone metabolism depending on the status of zinc in
the body.
In vitro studies have shown that zinc sulphate or I}-alanyl-L -histidino-zinc
inhibit osteoclast-like cell formation [9] and this as been related to the capacity
of zinc to stimulate bone formation in vitro and in vivo [19]. While a role for zinc
with copper and manganese has been proposed in osteoporosis [20], energy
dispersive X-ray (EDX) microanalysis and inductively coupled plasma optical
emission spectroscopy analyses of various metal ions in the cortical and iliac
bone of patients with osteoporosis has not shown evidence of subnormal zinc or
other metals [21]. This suggests that zinc deficiency is not a manifestation of
osteoporosis. It is possible that zinc supplementation has beneficial effects from
pharmacological actions of this metal ion although supplements of other metals
(copper, calcium and manganese) may contribute to the overall beneficial effects
in osteoporosis [20].
Inflammatory bowel diseases (IBD)

A substantial amount of information exists showing that subnormal levels of
zinc exist in the plasma of patients with colitis and Crohn's disease [22]. This is
probably a consequence of increased catabolic activity in these diseases with
concomitant renal loss of zinc [22]. As in rheumatoid arthritis and related
conditions, cytokines, especially those which regulate zinc metabolism and
metallothioneins, are probably responsible for the loss of zinc (as well as that
of copper and other metal ions of physiological importance)[22]. Thus, pro-
inflammatory cytokines, such as IL-l and TNF<X promote loss of zinc while IL-
6, considered by some to be anti-inflammatory, may negatively regulate this loss
through increased metallothionein synthesis, this being part of the acute-phase
response [22]. The subnormal levels of zinc may account for a partial immune-
deficient state in these individuals, e.g. reduced IL-2 by Thy-l lymphocytes [22].
Determining the extent of zinc deficiency and the value of monitoring this in
patients with IBDs has been subject of much debate [22]. The major issue is that
plasma zinc appears to be directly related to concentrations of albumin in the
circulation [22]. As with rheumatoid arthritis, the plasma albumin concentra-
tion is often reduced in patients with IBDs, probably as a consequence of the
catabolic effects ofIL-l and TNF<x-induced suppression of the liver synthesis of
this protein [22]. It appears that the capacity of albumin to bind zinc then
becomes reduced since this is the major plasma constituent binding zinc.
Recognition of the major role of albumin in regulating zinc status and the
technical difficulties in routine measurement ofleucocyte levels of zinc, probably
83
the most meaningful parameter clinically as well as biochemically, indicates that

determining circulating levels of this metal ion would not be of much value [22].
The clinician's approach to the treatment of zinc deficiency in IBD, therefore,
rests on the pragmatism of giving zinc supplements to patients with IBD,
together with an array of vitamins [22] including vitamin C [23]. Unfortunately,
this approach disguises assessment of the consequences of zinc deficiency since
the physician's desire is to see improvement in patients over all [22].
Chronic alcoholic liver disease and GI diseases

Zinc deficiency is manifest in alcoholic liver disease (ALD) by symptoms
including skin lesions, anorexia, taste abnormality, impaired vision, cognitive
dysfunction and hypogonadism [24]. Studies by McLain and others [24] have
focused on the role of the pro-inflammatory cytokines, IL-I, TNF", and IL-8, as
well as oxidative stress changes in mediating ALD-related Zn deficiency. Thus,
peripheral mononuclear production of these pro-inflammatory cytokines is
enhanced in patients with ALD as well as in animal model systems. The
intracellular transcription factor NFKB is also enhanced in monocytes of
patients and animals with ALD, partly as a consequence of oxidative stress.
In-vitro production of NFKB is reduced by addition of zinc ions to mono-
nuclear cells of ALD patients stimulated with endotoxin, and similar effects
have been ascribed to in-vivo zinc supplementation in subjects with ALD [24].
ALD induced in rats also leads to increased oxyradical and collagen
production and reduced metallothioneins, effects which are partially reversed
by feeding zinc [25].
These acute and chronic effects of alcohol on the development of zinc
deficiency and consequent effects on the pathogenesis of ALD may have
considerable relevance to understanding the mucosal changes and propensity
to gastrointestinal ulceration induced by alcohol.
'Subclinical' or primary zinc deficiency from diet and infections

Extensive studies have shown that zinc deficiency is evident in malnourished
individuals in less well-developed regions of the world as well as in western
countries [26-28]. Since Prasad et al. [26] described a syndrome confirmed later
by Halstead and co-workers [27] known as the Prasad-Halstead syndrome, in
growth-stunted adolescent children in Middle East countries, it has been
recognized that parasitic infections and geographia contribute to this syndrome
[28]. Major manifestations of zinc deficiency include retarded growth and
development, increased pregnancy complications, immune suppression, delayed
wound (skin) healing, dermatitis and impaired neuropsychiatric functions [28].
The Prasad-Halstead syndrome is associated with poverty and the consumption
of diets rich in unleavened wholemeal bread containing large amounts of
phytate, a powerful zinc chelator [28]. Meat, fish, poultry and milk, which are
sources of highly bioavailable zinc [29], are not readily consumed. Some
84
Western diets have been claimed to be deficient in zinc especially where there is
appreciable consumption of phytate [28]. Pregnant women seemed to be at
particular risk for zinc deficiency [28,29]. The consequences of this may be
important for the development of teratogenicity from inflammatory drugs since
it has been shown in rats that the teratogenic effects of salicylate are promoted
by zinc deficiency [30].
In addition to helminth, parasitic and bacterial infections producing zinc
deficiency [31-34], it is now evident that HIV infection is important in some of
the manifestations of CD4+ immune suppression in this condition [35].
Upper GI ulceration
Because of the importance of zinc as a factor in growth and repair of epithelial!
squamous tissue, much interest has been directed to the potential for zinc
compounds to prevent or repair ulcers of the gastroduodenal region [36] (see
also later section on zinc compounds as anti-ulcer agents). Several key enzymes
in the mucosa have requirements for zinc, including (a) carbonic anhydrase
isoenzyme II and other isoforms which regulate secretion of mucosal protective
bicarbonate ions and are co-secreted with mucus [37,38], (b) superoxide
dismutase which scavenges superoxide, (c) thymidine kinase, DNA and RNA
polymerases which are obviously important for nucleic acid synthesis, as well as
(d) other key enzymes and transcription factors involved in control of cell
growth [3].
In an endoscopic study of patients undergoing investigation for symptoms of
abdominal discomfort and pain, Kadakia et al. [39] found that serum zinc
concentrations were significantly lower in patients with endoscopically observed
oesophagitis than control subjects. Paradoxically, distal oesophageal tissue
concentrations of zinc were higher in the oesophagitis patients than controls. No
differences were found in the proximal or mid-oesophageal tissue zinc concentra-
tions between the two groups. In the patients that had oesophagitis and were
simultaneously receiving Hz-receptor antagonists, oesophageal tissue concentra-
tions of zinc approached normal values. The increased tissue concentrations of
zinc in the distal oesophagus of oesophagi tis patients are ascribed to rapid
proliferation in this region. The response to disease resembles that in synovial
tissue of patients with rheumatoid arthritis as noted earlier. The authors of the
oesophagitis studies [39] speculated that the increased tissue zinc concentrations
could be at the expense of those from the circulation. While this may occur, in
fact, it would seem unlikely to have great quantitative significance to the overall
decline in serum zinc as the tissue mass in the distal oesophagus taking up such
large amounts of zinc would be relatively small. It is more likely that the stress of
the disease state is responsible for the decline in circulating zinc.
Zinc deficiency in rats has extensive effects in reducing food intake, reducing
mucosal cell division, impaired intestinal disaccharidase activity, carbohydrate
and triglyceride absorption, increased amino acid loss and reduced mucosal
protein, concomitant with altered intestinal mucosal morphology [40]. In the
85
stomach, zinc deficiency increases gastric secretory volume, acid and pepsin and
promotes or aggravates stress-induced gastric lesions and decrease in mast cell
count [41]. Thus, it would appear that any state manifesting systemic zinc
deficiency will promote or potentiate the development of gastric mucosal
damage by increased mast cell histamine release and acid-pepsin secretion.
As indicated above, chronic alcohol intake and alcoholic liver disease
profoundly affects liver zinc homeostasis and this would be expected to have
indirect consequences for mucosal protection in the stomach since the liver is
such a critical regulator of stomach physiology. In rats, long-term ethanol intake
induces a zinc-deficient state due to increased faecal and urinary loss of zinc
[40]. Moreover, the metabolism of ethanol to acetaldehyde is impaired in zinc
deficiency [40]. Thus, in addition to creating a zinc-deficient state, alterations in
alcohol metabolism may have pronounced consequences for promoting acet-
aldehyde-induced mucosal and liver cell injury.
Alzheimer's and related dementi as

The role of zinc, like that of aluminium, in the manifestations of Alzheimer's
disease has been the subject of much interest, speculation and controversy [42].
While most studies have shown increased aluminium in the brains of Alzhei-
mer's patients, careful studies using inductively-coupled plasma source-mass
spectrometry have shown that hippocampal zinc is reduced in these subjects
[43]. Increased zinc has been observed in the nails of patients with Alzheimer's
disease [44]. The underlying mechanism and clinical significance of this
observation are not clear. Hepatic zinc concentrations have also been shown to
be increased in Alzheimer's disease patients with associated reduction in zinc
bound to metallothionein and increased binding to high-molecular-weight
proteins [45]. Plasma zinc concentrations were shown to be decreased in
Alzheimer's disease patients along with reduction in plasma levels of other
antioxidants, vitamins E, C and A, supporting the hypothesis that there may be
some relationship between antioxidant status and the development of this
disease [46].
Hypotheses about the role of zinc in Alzheimer's disease have been discussed
[42] and the situation is probably very complex and linked to the overall trace
metal, metabolic and inflammatory states in these individuals. One such
hypothesis suggests that the amyloid, which is abundant in specific regions
where neurofibrillary tangles are evident in the brains of Alzheimer's disease
patients, actually induces the formation of tangles [42]. An interesting aspect of
this theory is that lead may be responsible for the reduction in brain zinc [42].
Reduced neuronal superoxide dismutase from zinc deficiency may also con-
tribute to the ROS-induced tissue injury.
Extensive studies have shown that local inflammatory reactions are evident in
plaques or brains of patients with Alzheimer's disease [47]. Increases in
cytokines and acute-phase proteins have been reported in these subjects [47] as
well as increased brain concentrations of prostaglandin D2 and thromboxane
86
B2 , the former being especially elevated in the frontal cortex [48]. The potentially
beneficial effects of non-steroidal anti-inflammatory drugs have been explored
extensively and some positive results are evident [47,49], thus giving support to
the importance of inflammatory reactions in this disease.
One important pathological role of zinc in manifestations of Alzheimer's
disease might be in promoting aggregation of soluble amyloid ~A4 protein [50].
The cysteine-rich region of amyloid precursor protein (APP) is associated with
zinc binding and this binding of zinc alters the affinity of APP for heparin [51].
In view of the apparent deficiency in brain levels of zinc in Alzheimer's
patients [43] and the potential anti-inflammatory effects of this metal, it was
logical that a study of the effects of zinc supplementation should be undertaken
in such patients. Accordingly, Currie and co-workers [52] studied 5 patients
having NINCDS-ADRDA criteria for probable Alzheimer's disease who
underwent treatment with 440 mg zinc sulphate (100 mg elemental zinc) bd for
4 days. The patients showed a marked decline in cognitive functions and
saccadic eye movement performance accompanied by elevation, within 48 h,
of plasma amyloid precursor protein as well as plasma zinc, all of which
returned to pretreatment levels upon cessation of treatment. An extended
version of this study was planned but was terminated after the results with the
5 patients showed the marked decline in cognitive function upon zinc treatment.
These observations are of potential interest but it should be pointed out that
the authors only studied the effects of what is a rapidly absorbed soluble zinc
compound. Slow-release forms ofzinc may produce different results. Interpreta-
tion of these results leads to several questions:
(a) Is the dose of zinc, which is sufficient to cause elevation of plasma levels,
too high for these subjects?
(b) Is there a pro-inflammatory or pro-immunoregulatory effect of zinc in

these patients?
(c) What relationship has the intake of zinc to the copper status in these
subjects?
(d) What relationship exists between the effects of zinc on plasma APP and
the clearance of this in Alzheimer's disease patients?
(e) Is a zinc challenge test one way for diagnosing Alzheimer's disease?
(f) Will zinc-chelating agents be useful in treating Alzheimer's disease?
(g) Is the apparent therapeutic benefit of NSAIDs in Alzheimer's disease

related to effects on zinc status?
These and other questions raise important issues for the utility of zinc
supplements in Alzheimer's disease as well as for understanding the apparent
pathological consequences of zinc in this state.
87
ZINC THERAPY IN INFLAMMATORY AND GASTROINTESTINAL

DISEASES
The above-mentioned studies on the apparent negative effects of zinc supple-
mentation in Alzheimer's disease are an important background for evaluation
the effects of zinc compounds in the therapy of other conditions. In studying the
effects of zinc compounds in inflammatory and gastrointestinal diseases, we are
considering tests of hypotheses about the role of zinc in the cellular pathology of
these conditions as well as testing whether the zinc compounds have any
therapeutic benefits.
Rheumatoid arthritis
Simkin [8] was the first to report the effects of zinc supplementation on the
progress of patents with rheumatoid arthritis. He based the hypothesis for this
trial on observations of the beneficial effects of oral zinc sulphate on chronic leg
ulcers of sickle cell anaemia, a condition complicated by acquired zinc
deficiency. He reasoned that, since leg ulcers are a manifestation of rheumatoid
disease a trial of oral zinc for this complication seemed appropriate. Further-
more, the zinc deficiency in this disease was another reason to study the effects
of zinc treatment. Also, a favourable response to a pilot study of zinc treatment
in a patient with rheumatoid arthritis, who had leg ulcers was so good that
'there was a moral obligation to conduct a double-blind study in rheumatoid
patients whose disease remained active despite partial suppression by conven-
tional therapeutic agents' [8]. Thus, the study involved observing the effects of
250 mg zinc sulphate hepatohydrate (50 mg elemental zinc) in capsule form
given three times daily with meals for 12 weeks in a double-blind cross-over (to
placebo) trial in 24 patients. Evaluations included grip strength, time to walk 50
feet, sum of points for swelling and tenderness in 68 joints, patients' 'global'
assessment of their condition, severity of morning stiffness, Westergreen
sedimentation rate, and various laboratory parameters determined initially then
at 2- and 4-week intervals. All the clinical parameters improved in the ZnS04-
treated groups and the mean serum zinc rose from 841lg/dl to 1161lg/dl. Serum
histidine declined from 1.57 to 1.36 mg/dl. Upon cessation of therapy, most
patients considered the disease had worsened without zinc and improved upon
reinstitution of zinc therapy. While no patients were considered to have been
cured, many were obviously better with zinc treatment.
Several other authors have reported studying the effects of zinc compounds in
treating rheumatoid arthritis, and the results obtained have been rather variable
[8] (see also review by Fernandez-Madrid in this book, Chapter 8). As pointed
out by Fernandez Madrid (Chapter 8), methodological issues as well as the zinc
status and clinical conditions of the patients will, in all probability, underlie the
likely response to oral zinc therapy.
88
Topical zinc in arthritis

Locally administered drugs applied to the inflamed region is probably the most
satisfactory means of therapy, providing, of course, there are no systemic effects
of the drug. Topically applied drugs have the advantage of reducing the burden
oftoxic effects on the gastrointestinal (GI) tract (if taken orally) and metabolism
by the liver. Furthermore, topical application of drugs has the advantage of
being a controllable means of drug delivery [53]. This is particularly the case for
lipophilic metal complexes [53]. Fairlie and Whitehouse [53] have considered the
theoretical and practical issues of a number of metal complexes applied as
transdermal systems in animal model systems.
Among the most interesting of the lipophilic metal complexes which, when
applied to the skin, has been found to be effective in controlling polyarthritis is
zinc monoglycerolate [ZMG; Glyzinc; Glyzinc Pharmaceuticals Ltd., now
owned by Pharmaction Ltd., Melbourne, Australia]. The preparation, physical
properties and crystalline morphology of ZMG have been previously described
[54,55]. The development of ZMG arose from extensive investigations by Dr
R.M. Taylor (of the Division of Soils, Commonwealth Scientific and Industrial
Research Organization, Adelaide, Australia) in which slow-release trace-metal
complexes were developed to serve as a controlled release source of trace metals
to overcome deficiencies of these ions in the soil. While this potential was never
realized, the development of ZMG as an antiarthritic and antiulcer agent has
attracted much interest. This lipophilic, highly lubricious, polymeric slow-
releasing zinc complex has been found to be highly effective when applied
topically or subcutaneously, e.g. in DMSO/glycerol mixtures, or rubbed on to
the skin as a powder in therapeutic or prophylactic models of treatment for
controlling fore- and hind-paw swelling of adjuvant-arthritic rats [56]. ZMG
was ineffective in controlling this disease when given orally and also, when
applied subcutaneously, was not effective in controlling the acute paw inflam-
mation induced by subplantar injection of carrageenan to rats (56).
Topically applied ZMG is non-irritant to the skin and, indeed, when
administered subcutaneously (in DMSO/glycerol) does not elicit inflammatory
responses even though there is a localized encapsulation at the injection site [56].
The antiarthritic effect of ZMG observed in rats, when given as a prophylactic
treatment regime, is akin to that observed with immunoregulatory agents (e.g.
cyclosporin A, cyclophosphamide) and indicates that there is a component of
the antiarthritic actions of ZMG which can be considered a consequence of the
immunoregulatory activity of zinc. While conventional zinc oxide (which of
course is used in skin ointments) exhibits some anti-inflammatory effects in
adjuvant arthritic rats, this compound is more difficult to apply than ZMG, and
the effects are less impressive and more erratic than ZMG (Rainsford and
Whitehouse, unpublished studies).
The antiarthritic effect of ZMG is evident in the reduction of soft-tissue
swelling and the periosteotic reaction in the tibiotarsal joints of the hind limbs
of arthritic rats [56] where the mycobacterial adjuvant exhibits most profound
89
joint inflammation with subsequent cartilage and bone destruction.
Acute anti-inflammatory activity

Intraperitoneal zinc chloride (in saline) has been found to reduce carrageenan-
induced pleurisy in rats as evidenced by reduction in pleural exudation and
neutrophil accumulation [57]. In contrast, the lack of effect of ZMG on hind-
paw inflammation induced by carrageenan [56] may be due to insufficient zinc
ions reaching the inflamed site. In studies by Yatsuyanagi et al. [57], the injection
of zinc into the peritoneal cavity would presumably have enabled this ion to
move rapidly into the pleural cavity where the inflammation was induced. This
twin-compartment model of inflammation separated by the well-vascularized
smooth muscle of the diaphragm is a useful means of examining the direct
functions of zinc and indeed other agents, in vivo without the confounding
problem of the putative anti-inflammatory agent coming into direct contact
with the inflammagen. Other investigations by Yatsuyanagi et al. [57] showed
that zinc chloride ip (16.4 mg Zn/kg) markedly reduced neutrophil chemotaxis,
phagocytosis and superoxide production in the pleural inflammation model in
vivo; these effects being corroborated from studies in vitro in which these
neutrophil responses from rats were shown to be significantly inhibited by 100-
300 IlmollL zinc [57]. Thus, control of both the vascular permeability and
neutrophil accumulation/activation by zinc may account in part for the acute
anti-inflammatory properties of this metal ion. Other cellular and immunologi-
cal effects of zinc are considered below.
Topical anti-inflammatory and antimicrobial effects and wound healing

The application of zinc oxide and other zinc-containing preparations for wound
healing and anti-inflammatory effects in skin conditions is legendary [58, 59].
Zinc undecylenate combines the anti-inflammatory effects of zinc with the
fungistatic and possibly fungicidal properties of undecylenic acid [59].
Antimicrobial activity is also exhibited by other zinc compounds [60], and
this may partly account for the benefits ofzinc compounds in treating acne [61-
63]. Experimentally induced wounds in rats are healed by zinc but this depends
on the localization of the wound [64]. Zinc is depleted in the serum, livers and
hearts of rats with induction of wounds but elevated in the serum, liver, kidney
and spleen as well as the wound site upon application of zinc-containing tape
[64]. This and other evidence [65] shows that there is absorption of zinc across
the skin in laboratory animals. In humans, application of zinc oxide ointment to
the skin does not lead to an appreciable increase in serum zinc [66]. This
probably reflects a high safety margin for topical zinc ointments since the
dynamics of release from cutaneous depots accompanied by ligation with thiol
compounds, with subsequent distribution and elimination are such that these
are all appreciably time-dependent processes. Thus, topically applied zinc
compounds can be safely employed for treatment of a variety of local
90
inflammatory conditions, including skin infections.

While there may be variable control of skin bacterial infections by zinc
compounds [67, 68], the major effect of topical zinc compounds may be the
local control of inflammation as well as inducing microbial stasis [60, 63]. There
is also possible impairment of interactions between cells and certain viruses (e.g.
herpes simplex [69]) and stimulation of local skin immunity helping to achieve
control of infection-associated inflammation.
The variable response in wound healing ascribed to zinc [64] may be
improved by the design of novel zinc complexes (e.g. zinc sulphadiazines for
burns [70]).
Antiulcer effects
General aspects of the antiulcer effects of zinc compouds has been reviewed
recently [36]. Two zinc complexes have been studied extensively for their anti-ulcer
activity in humans, namely zinc acexamate (Laboratorios Vinas S.A., Barcelona,
Spain) and zinc carnosine (Polaprezinc, Z-103; Zeria Pharmaceutical Co. Ltd.,
Tokyo, and Hamari Chemicals Ltd., Osaka, Japan). Zinc sulphate was found in
earlier studies [71,72] to have ulcer healing activity in gastric ulcer disease.
However, zinc sulphate is a potent astringent and can cause oesophageal irritation
and pain. The development of zinc complexes has, therefore, been designed to
overcome these untoward irritant effects of free zinc ions and at the same time
achieve some control over the absorption of zinc from the intestinal tract.
Recently, complexes of zinc with the Hrreceptor antagonists, cimetidine and
ranitidine, have been studied for their antiulcer activity [73]. A considerable
number ofpatents exist claiming anti-ulcer activity of zinc-containing compounds.
Zinc acexamate
By far the most substantial data published on the anti-ulcer effects of a zinc
complex in humans has come from studies with zinc acexamate (ZAC) [74-76].
Thus, in a multicentre study of 276 patients with rheumatoid arthritis in Spain
who were treated with the NSAIDs, diclofenac, piroxicam, naproxen or
ketoprofen, it was found that 300 mg ZAC nocte significantly reduced the
endoscopically observed gastroduodenal mucosal damage after 28 days treat-
ment, compared with placebo taken under the same conditions [75]. Allowing for
26 patients who withdrew from the trial and 41 who were lost to follow up, it was
found that after treatment with ZAC, 88% had normal endoscopically observed
mucosa while 66% of those who received placebo had a normal mucosa [75]. The
relatively high placebo rate was not surprising since there is an appreciable
mucosal adaptation evident after 3-4 weeks treatment with most NSAIDs
[77,78]. There were no side-effects observed after treatment with ZAC [75].
ZAC was originally investigated for efficacy in treating peptic ulcer disease
[74]. In the earlier smaller scale studies in the 1980s ZAC was found to be
equiactive with the Hrreceptor antagonists, cimetidine and ranitidine, in the
91
rates of healing of gastroduodenal ulcers [74]. The incidence of side-effects in the

ZAC treated group was low [74].
A meta-analysis of all randomized controlled clinical trials performed with
ZAC was reported by Jimenez et al. in 1992 [76]. Of 18 studies, 13 of these
comprised investigations in 757 patients that were considered in the final
analysis. The trials appear to have been conducted in Spanish centres and most
of the original investigations were reported in Spanish medical journals. The
duration of treatment ranged from 3-6 weeks and the daily dose in most of these
trials was 900 mg/d ZAC, although one of the studies was conducted with 300
mg/d ZAC and, in another, some patients had doses up to 1800 mg/d [74]. Most
of the patients were in the 40-60-year-old group and there was a preponderance
of males in most of the trials. Three of the studies were compared with placebo,
three with cimetidine 1000 mg/d, six with ranitidine 300 mg/d and one with
famotidine 40 mg/d.
The results of this analysis showed that ZAC was (a) superior to placebo
(pooled odds ratio 5.55) and (b) not different from the Hrreceptor antagonists
in patients with gastric, duodenal or peptic ulcers [74]. A low frequency of side-
effects ( < 3%) appeared in all treatment groups, including placebo. The side-
effects reported were nausea, vomiting, constipation, somnolescence, headache
and dry mouth [74]. This is the largest group analysis of anti-ulcer trials
reported with any zinc compound. The lack of any appreciable side-effects and
comparability in effect with established Hrreceptor antagonists is encouraging.
The protective effects of ZAC have been shown in a range of animal model
systems and the mode of action defined in part [79-85]. Most of the actions of
ZAC may be related to the actions of zinc ions released from the complex with
acexamic acid.
Table 1 gives a summary of the actions of zinc against gastric mucosal
damage induced by experimental ulcerogens [36,79-96,98,99,102-111] together
with concepts from non-mucosal systems that may have importance in the
actions of zinc in preventing mucosal damage [97,100,101,112]. It is apparent
that the antiulcer activity of zinc compounds can be considered to have a
multifactorial basis. While this may offer advantages, especially since most
upper GI ulcers involve multiple mechanisms in their aetiology it would be
useful to be able to identify which of the various protective actions of zinc is
important at different stages in the development of gastric injury.
Zinc carnosine
Zinc carnosine has been shown to have antigastric-ulcer activity in rat models
[10-107] and this has been related to to its antioxidant activity [102-105]. Part of
the antioxidant effects of zinc carnosine may be due to this compound
stimulating the synthesis or activity of superoxide dismutase and glutathione
peroxidase [105]. Unpublished reports (Hamari Chemicals Ltd., Osaka, Japan
to KDR) exist claiming that zinc carnosine inhibits growth of Helicobacter
pylori.
92
Table 1 Multiple mechanisms of antiulcer activity of zinc compounds
1. Secretory Functions
Reduced output of acid and pepsin.
- Reduction in histamine - partly related to stabilisation of mast cells
- Increased mucus production
- Physicochemical stabilization of mucus molecules?
- Effects on actions of histamine
2. Mucosal membrane 'stabilization'

Stabilization of lysosomes
3. Eicosanoid metabolism
Increased prostaglandin E production
- Reversal of enhanced leukotriene production
4. Vascular dynamics
Increased blood flow
- ?Protection of microvessels in mucosa
5. Leucocyte functions
Inhibition of oxyradical production*
- Reduced enzyme release·
- Enhanced phagocytosis*
6. Sulphydryl reactivity
Reduced in protein(s) of mucosa
- Altered glutathione status
- ?Relevance- reduced activity of metalloproteinases
7. Prevention of energy metabolic changes
8. Prevention of cell-programmed death, i.e. apoptosis*
9. Irreversible inhibition of growth of Helicobacter pylori
• Not demonstrated in gastric systems

Based on References 36, 57, 79-105,108-112,139
Reproduced with permission of the publisher, Kluwer Academic Publishers
The in-vitro uptake and transport of zinc carnosine has been studied using the
rat everted intestinal sac technique [106]. The transport of zinc carnosine was
found to exhibit Michaelis-Menten kinetics and was energy dependent. These
studies indicate that the transport of zinc carnosine is a carrier-mediated process
dependent upon the activity of Na+, K+-ATPase [106]. In all probability the
zinc(II) from zinc carnosine is first slowly released from the complex and then
actively transported across the mucosal cell membrane.
In-vitro cytoprotective effects of zinc carnosine have been demonstrated
93
against ethanol-induced damage to the cells of rabbit gastric glands [107]. Thus,
the injury from 8% ethanol (measured by release of lactate dehydrogenase) was
reduced by prior incubation with zinc L -carnosine but not L -carnosine alone,
showing that the cytoprotection is due to the zinc in this complex [107].
Zinc monog/ycero/ate (ZMG)

This polymeric complex probably has quite different physicochemical properties
compared with ZAC and zinc carnosine. This may be important in determining
its antiulcer properties. Thus, the pH-dependent dissociation of Zn 2 + from
ZMG and the competitive association with endogenous ligands [55] probably
has considerable importance in determining the amount of bioactive Zn 2+
which is available for exerting protective efffects in the GI mucosa. At low pH,
the release of Zn 2 + from ZMG is greater than at neutral pH. Thus, in the
stomach, the acidic milieu is favourable for dissociation of Zn 2+. Since acidity in
the stomach is a major factor in ulcerogenesis, this will promote conditions in
which Zn2 + is released from ZMG. Clearly, the amount released is not like that
from ZnS04 or other salts. Hence, the irritant responses which with ZnS04 do
not arise with ZMG. These theoretical issues and observations of the low level of
mucosal irritancy due to ZMG compared with the profound mucosal irritancy
from ZnS04 has been established by scanning electron microscopic observa-
tions in rodents given oral doses of these compounds [108].
Orally administered ZMG exhibits dose-related antiulcer effects in rodents
against gastric mucosal damage induced by NSAIDs, ethanol with or without
added HCI, and reserpine [109]. ZMG also inhibits the intestinal ulceration
induced by indomethacin given either orally or subcutaneously to rats [l08].
The mechanisms of protective effects of ZMG against NSAID-induced
mucosal damage probably involve normalization of the perturbed eicosanoid
metabolism induced by these drugs. Thus, inhibition of mucosal prostaglandin
E2 (PG) by aspirin (200 mg/kg po) is reversed in mice by ZMG (50 mg/kg po)
given 4 min prior to aspirin [l08]. Also, the increase of 5-lipoxygenase activity
induced in mice under the same conditions of aspirin dosage is reversed by the
same dose of ZMG [108]. Thus, the depletion by aspirin of mucosal protective
PGs and the concommitant excess production of vasoconstrictorlleucoattrac-
tant leukotrienes, is reversed and so normalized by ZMG [108]. The release of
Zn 2 + from ZMG probably also causes other cellular and molecular actions, as
noted in Table 1.
ZMG, like Zn 2+ [110] and zinc carnosine [unpublished studies by KDR] has
been found to inhibit the growth and viability of Hpylori but we found this only
when these compounds were combined with the complexing agent, ~-cyclodex
trin [110]. More pronounced inhibition of H pylori was observed when the
culture medium was pH 5.0 rather than pH 7.2. This may reflect the more
pronounced dissociation of Zn 2 + from the ~-cyclodextrin complex as well as
effects on growth of H pylori at these two different pH conditions.
94
Other compounds
As indicated previously, zinc--cimetidine has been shown to have ulcer-healing
effects in acetic acid-induced ulcers in rats [73]. The model employed involved
injection of 0.05 m1 20% acetic acid into the submucosal layer at the junction
between the fundus and antrum and the test substances were given orally twice
daily for 14 days followed, on the 15th day, by measurements of the ulcer size
and other parameters. A dose-related reduction (15-60 mg/kg po) in ulcer index
was observed with zinc cimetidine but not with the molar equivalent of zinc as
zinc chloride [73]. The index of mucosal regeneration was increased with both
the complex and the mixture of cimetidine and zinc. The ulcer-healing effect of
zinc cimetidine was preceded by an increase in thiobarbituric acid reactants,
sugggesting that an antioxidant effect of the complex was responsible for this
ulcer healing activity. Oddly, the zinc--cimetidine complex did not inhibit acid
secretion even though cimetidine did, so it is puzzling to understand why this
complex was developed in the first place. This is especially so as the
antisecretory effect of cimetidine would have been expected to have a specific
advantage in being incorporated in the complex.
The zinc-amino acid complexes, zinc-aspartate and zinc-glycinate, given
intraperitoneally reduced the incidence, number and severity of reserpine-
induced ulcers in rats [111]. This was paralleled by histochemically observed
increases in PAS+ve mucosubstances in the outer mucous cells, decrease in
RNA in Chief cells, and increased periglandular capillary ATPase, the latter
being interpreted as reflecting improved gastric mucosal microcirculation [111].
These observations may reflect the actions of endogenous zinc-amino acid
complexes following the oral administration of zinc compounds.
Inflammatory cell and immunological effects

A considerable amount of work has been published on the effects of zinc
deficiency on the functions of the immune system [see References 4, 22, 33, 34,
112-115]. Zinc deficiency may result in direct actions of cells of the immune
system and indirect effects that influence the immune functions by primary
alterations in other systems (e.g. the neuroendocrine axis [116]). As indicated
previously, a tacit deficiency in zinc may occur as a result of infection,
inflammatory reactions or the development of gastrointestinal ulceration or
diseases. Direct and indirect influences of these zinc-deficient states may,
therefore, affect immune functions. Zinc deficiency can produce increased
susceptibility to a number of pathogens [113, 114, 117] although the toxins from
bacterial pathogens may lead to increased zinc absorption from the GI tract
[113]. Both the innate and aquired immune systems are influenced by zinc
deficiency at the level of mucosal, barrier and mobile cellular systems [114].
Involution of the thymus, leucopaenia with accompanying growth retardation
and weight loss are generalized responses to zinc deficiency [114]. There may be
alterations in T-cell subsets with zinc deficiency although this does not always
95
occur in all experimental systems [114]. Uniform reduction in T-cell numbers is

generally observed. Delayed-type hypersensitivity reactions are attenuated in
zinc-deficient animals [1l3, 114], reflecting the reduction in T-cel1 numbers. In
most zinc-deficient animal models the responses to infection or macrophage
function are restored to normal by addition ofzinc supplements either in vivo or
in vitro, thus showing that the effects of zinc on these components of the
immune system are direct and not a consequence of anorexia, neuroendocrine
abnormalities, weight loss or other indirect physiopathological effects.
As with deficiencies in other micronutrients, the induction of zinc deficiency
can attenuate specific autoimmune states although any al1eged beneficial
influences must be considered marginal [114], the extensive systemic manifesta-
tions of zinc deficiency being such as to outweigh any benefits.
In humans, total parenteral nutrition often leads to zinc deficiency and
accompanying impairment of immune functions, among them leucopaenia,
depressed T-cell mitogenic responses, decreased NK activity, increased CD4+
suppressor and decreased CD8+ helper T-cell populations [114]. Loss of
immunological responses with aging is now well recognized [114, 116] and they
may have various origins, among them decline in neuroendocrine function [116]
and increase in apoptosis, the latter being physiological1y regulated by zinc
[118]. An appreciable component of the effects of zinc on the components of the
immune system may be as a consequence of decline in cytokine production or
actions (e.g. interleukin-2) [114] (see later).
At the other extreme the ingestion of large amounts (mega-doses) of zinc
supplements can paradoxically also lead to profound immune-deficient states,
including reduction in antibody production, T- and B-cel1 mitogenic responses,
natural kil1er (NK) activity and neutrophil and macrophage functions [115].
Caution has, therefore, been expressed in the use of zinc supplements [115].
Even doses as low as 300 mg daily have been found to cause reversible
depression in T-cel1 and neutrophil functions in young healthy men consuming
zinc supplements for 6 weeks [115]. The view that oral zinc supplements are safe
is to be modulated in view of these concerns.
Zinc also corrects the immune function depressed by tumour burdens [119].
Thus, zinc acetate 3 mg/kg x 2 days ip increased the survival time in mice given
Ehrlich's ascites tumour [119]. By 30 days post-challenge, 88% of treated
animals survived compared with 49% of control animals and 49% of those given
a single dose of cyclophosphamide 75 mg/kg ip [119]. There are multiple effects
accounting for the antitumour effects of zinc [120,121], aside from the
restoration/stimulation of immune surveillence.
Several studies have reported development of zinc derivatives with potential
antitumour activity [122-127], including some amino acid complexes [122].
Some of these studies show direct inhibition by zinc compounds of tumour
growth in vitro.
The reported antiviral effects of zinc compounds [128-l33] may also be due to
direct actions on the growth of viruses [128-l30] as wel1 as stimulation of
immune surveillence [121, l31]. The practical utility of zinc compounds may
96
reside in the prevention or treatment of rhinovirus colds and herpetic infections

[132,133].
Cellular immune effects

Tables 2-4 summarize the cellular immune responses by exogenous zinc
compounds in model systems in vitro and in vivo in animals. These responses
can be divided into those which influence (a) the phagocytic activity of
neutrophils (Tables 2 and 3), (b) the accumulation and activation of neutrophils
(Table 3), (c) the activation of mast cells (Table 2), (d) lipid oxidation from
reactive oxygen species (ROS) released by neutrophils (Table 3), and (e) the
release of cytokines and intracellular reactions in lymphocytes (Table 4).
It is well established that zinc compounds stabilize lysosomes in cells against
the labilizing influences of various exogenous agents and pathogens (Table 2).
This influences the phagocytic capacity by neutrophils as well as lytic activity in
non-phagocytosing cells. The well-known inhibitory effects against mast cell
degradation is another effect of zinc ions which protects against release of
histamine (Table 2). Reduced production of histamine has been observed in the
gastric mucosa of rodents in vivo and, while this effect may be important for
gastric ulcerogenesis in these species, it is not known if this is paralleled in
humans, e.g. in gastric/peptic ulcer disease or asthmatic conditions.
While several studies have convincingly demonstrated that zinc ions in the
10-150 ~mol!L concentration range inhibit phagocytosis with concomitant
reduction in the release of oxyradicals other studies have demonstrated basal
production of superoxide and hydrogen peroxide by 1-100 ~ol!L zinc ions
(Table 3). This increase in ROS is apparently due to a G-protein coupled system
being activated by zinc. It is not clear from these studies whether zinc was
exhibiting mild cytotoxic responses, especially at the higher concentrations,
which in turn were responsible for the activation of neutrophils to produce ROS.
In other studies, NADH oxidase was found to be inhibited by zinc ions and,
while the bacterial system in which this was observed may not be identical to
that in neutrophils, which is coupled to the ROS-generating system, it is possible
that this might represent another site of action of zinc on the ROS system.
Blockade or activation responses on channels in cells is a mechanism of zinc
on nerves [134-136] and smooth muscle [137]. Inhibition of intracellular
calmodulin functions is another reported action of zinc (Table 3) and so both
calcium-channel and Ca2 +-dependent intracellular mechanisms, some of whose
functions are important for leucocytes, could be important sites of action in the
latter cells accounting for the effects of zinc on inflammatory and immunologi-
cal systems.
Recently, there has been much interest in the potential for zinc to modulate
the production or even actions of pro-inflammatory cytokines, particularly
interleukin-l and tumour necrosis factor-ex. (Table 4). The stimulation by zinc
ions of IL-l~ and TNFex. has been shown to occur in peripheral blood
mononuclear cells (PBMC) but the quantitative effects of this metal ion are
97
Table 2 Effects of Zn Compounds on Lysosomal Enzyme Release and Mast Cell Degranulation (")
0
1]
1]
m
Concentration Rangel Pharmacological ;U
:l>
Compound Effects EC50 consequences References z
0
N
Z
(")
Zn 2 + (various Stabilization of isolated rat 50-100 j.lmollL Prevention of cell autolysis 142-144
compounds) lysosomes and tissue destruction by
Z
Z
"T1
lysosomal enzymes
>
;s::
ZnS04 Stabilization oflysosomes 10-11-10-2 j.lmollL Protection against mucosal 92 ;s::
(0 peak at 10-2 j.lmollL damage ~
0
CO
~
Zinc acexamate Inhibition of Triton X-IOO 0.2 j.lmollL Protection against lysosomal 82 :l>
z
induced labilization of damage of degranulation 0
0
lysosomes of mucosal mast cells m
G>
m
z
m
Zn2 + Protection against histamine 4 mg Zn2+ /d In-vivo inhibition of mast 92
release in vivo in rat lungs cell degranulation ~<:
induced by reserpine m
g
en
~
m
en
Table 3 Effects of Zn compounds on oxygen consumption, ROS and lipid peroxidation
Concentration Rangel Pharmacological

Compound Effects EC50 consequences References
ZnS04 Inhibition in vivo of mobiJizaiton and phago- 5.0m/kgbdx3d Inhibition of recruitment 112
cytosis by PMNs in rat peritoneal cavity in rats and activation of PMNs
Zn2+ Inhibition of latex-activated oxygen uptake 6.15-153.0 ILmollL Inhibition of PMN activation 112
(form not stated) by dog PMNs but not by resting cells ECso = 11 0 lLffiollL
ZnCl z Inhibition of rat neutrophil chemotaxis (1) ECso = 133 lLffiol/L Inhibition of recruitment 57 N
Z
(into pleural cavity (l), phagocytosis (2) (2) EC so = 150 lLffiollL and activation of PMNs ()
and Oi --release (3) (3) EC so =60 ILmollL Z

Z
c.o ."
c.o ZnS04 Decreased luminol chemiluminescence in 1st (1) 1-1000 ILmoi/L Effects on Oi - and RzO z by 146 >
;::
peak and increased in unstimulated (1) rat (2) 100 lLffiol/L G-protein dependent system ;::
peritoneal PMNs while fMLP induced ~
increased (2). First peak inhibited by SOD 0
z
or catalase. Zn2+ induced Oi.--inhibited by
pertusis toxin but not PKC inhibitor or
calmodulin antagonist
ZnS04 Inhibition of succinic oxidase from 10-100 ILmollL Blockage of oxidative metabolism 147
E. coli reversed by ~mercaptoethanol
(ME). Inhibition ofNADR oxidase
not affected by ME
Zn2+ Inhibition of calmodulin function EC so =40-50 lLffiollL 148

(activation of Ca-ATPase in RBCs)
Table 4 Effects of zinc compounds on mononuclear cell/cytokine reactions ()
o
~
m
Concentration Rangel Pharmacological ;;0
Compound Effects ECso consequences References »z

o
~
z
()
ZnCl 4 Stimulation ofTNFcx and IL-I~ production 0.2.0 mmollL peak at Stimulation oflymphocyte 149
by human PBMC in presence but not absence 250 ~mol/L responses Z
Z
of FCS and endotoxin. No effect on IL-6 "T1
production. Increased TNFcx related to ~

increase in TNFcx-mRNA s::
-"
o~
8 ZnS04 Increased TNFcx and IL-I ~ by human PBMB 12.5-100 ~mol/L Stimulation of lymphocyte IL-l 150 ~
with LPS. Effect on TNFcx>IL-I~. Same effect and TNFcx. Selective effects on ~
in whole blood assays. MHC-II, cf LPS-mediated o
o
Increased IL-I ~ with bacterial superantigens 12.5-100 ~ollL pathways of lymphocyte activation
~
SEA, SEE and MAS, with LPS but not PHA or m
z
m
TSST-l.
Decreased IL-l ~ from whole blood and PBMN ~
with SEA, SEE and MAS, but not TSST-I <:
m
without LPS. Decreased TNFcx in whole blood g
(J)
and PBMN with SEA and SEE alone
~m
(J)
Table 4 (cont).
Concentration Rangel Pharmacological

Compound Effects EC50 consequences References
N
Znso 4 Zn2+ effect on IL-1 ~ production by PBMN 100--500 Ilmo1/L 151 Z
(")
dependent on protein composition of media
Z
and presence of transport proteins (e.g. Z
->- "T1
0 transferrin - which stimulates uptake of Zn ,....
->- in 1eucocytes) ~
s::
ZnC1 2 Stimulation of lymphocyte proliferation by 1.5-6.0 llffiol/L Stimulation of T-cells 145, 152 ~
6
alleviating PGE2 inhibition of proliferation z
Zn2+ (salt Stimulation of tyrosine phosphorylation of 1-1000 llffiollL Activation of T-cells 5

not stated) p56lck protein in LSTRA murine lymphoma
cells
dependent on the nature of the stimulating agent that is employed (Table 4).
Increased TNFrx production seems to be a predominant effect of zinc ions and
this appears to be a consequence of an increase in the mRNA that codes for
TNFrx (Table 4), suggesting that this ion may be influencing transcriptional
responses specific for the production of TNFo:.
Another intracellular site for the modulating actions of zinc that maybe
important in cellular regulation is the tyrosine phosphorylation system which
is stimulated by zinc ions (Table 4). This may have importance for the regulation
of actions of other cytokines.
Prostaglandin E2 production is stimulated by zinc ions [93] and this, together
with inhibitory effects on LTB4 production [101], may affect leucocyte functions
(Table 4). Counteracting this is the enhancement of T-cell proliferation by zinc
which overcomes the inhibiting effects of PGE2 on lymphocyte proliferation
(Table 4).
Overall, zinc ions would appear to have immunostimulant activities, particu-
larly those involving thymocyte and mature T-cell proliferation and the
production of pro-inflammatory cytokines. This is contrasted with the acute
anti-inflammatory effects of zinc manifest at the level of (a) inhibition of
neutrophil accumulation at inflamed sites and subsequent activation, and (b)
mast cell activating reactions.
The immunostimulatory effects from production of proinflammatory cyto-
kines maybe of considerable significance in the antitumour actions of this metal.
Less clear is the role of immune stimulation in the potential antiarthritic activity
of zinc. While there maybe subtle, as yet undefined, alterations in the ratios ofT-
suppressor CD4+ to T-helper CD8+ cells as well as switch of Thl /Th2 helper
subsets [140] by zinc that contribute to its antiarthritic effects, this may not
alone explain why production of pro-inflammatory cytokines by zinc should
lead to its antiarthritic activity.
The acute anti-inflammatory effects of zinc appear to be limited to responses
of neutrophils and mast cells. The paradoxical increases in PGE2 production by
zinc may limit the effectiveness of this metal in achieving marked anti-
inflammatory responses. Certainly, the inhibition of ROS production in
neutrophils and other systems by zinc is of major significance, especially for
the reported anti-oxidant actions of this metal ion [138,139].
The extent to which the role of exogenous zinc is in the modulation of the
actions of those zinc-finger proteins that are important for regulating the
production and/or actions of cytokines or other cell-regulatory event [140], is
not yet known.
Zinc in apoptosis
The precise role for zinc in apoptosis [100,1 18,141,162-165] has not been well
defined, with some studies outwardly conflicting. Evidence has been presented
that increasing cellular zinc content inhibits colchicine-induced apoptosis [162].
In this study, zinc was administered to cells in the presence of an ionophore,
102
pyrithione, which allowed far greater concentrations of zinc to enter the cell
than with zinc alone. The added zinc was visualized with the zinc-specific
fiuorophore, Zinquin, which allowed the measurement of changes in the small
free pool of zinc within the cytoplasm. Apoptosis, as measured by DNA
fragmentation assay, was inhibited directly proportionally to increased levels
of the labile zinc down to a concentration of 5 ~mol!L zinc in the culture
medium. Use of a zinc-specific chelator, TPEN (NNN'N'-tetrakis-(2-pyridyl-
methyl)ethylenediamine), abolished the inhibition of apoptosis mediated by zinc
in a concentration-dependent manner. It should be noted with caution that the
use of the ionophore to artificially increase the labile zinc pool may not be
directly applicable to the uptake of zinc in a physiological sense but does
illustrate a possible role for the labile zinc pool.
In contrast to a proposed inhibitory role of zinc in apoptosis are observations
that have shown a subset of murine thymocytes for which zinc is an inducer of
apoptosis [141]. Zinc, at concentrations of 80-200 ~mol!L, caused CD4+CD8+
cx~TCR10CD3&lo to undergo apoptosis and also to a lesser degree the single
positive CD4+ or CD8+ subclasses. The concentration range within which zinc
has this effect is outside the normal physiological range but it is postulated in the
study that the intracellular levels for the thymocytes do not rise much above 1
~mol/L when incubated with 100 ~ol/L zinc ions.
Other studies have shown that, in vivo, zinc may have no direct effect on the
reduction in lymphocyte numbers by apoptotic depletion [163]. These studies
showed that mice fed a zinc-deficient diet develop lymphopoaenia and thymic
atrophy but that splenocytes recovered from these mice show normal class and
subclass phenotypes and normal response to mitogen. Additionally, macro-
phages isolated from these mice recovered most of the normal functions when
incubated in the presence zinc ions. However, adrenalectomy of mice fed a zinc-
deficient diet protected them from B-cell depletion and thymic atrophy. In
contrast, administration of a glucocorticoid, in concentrations equivalent to
those found in mice fed a zinc-deficient diet, to mice fed a normal diet induced
B-cell depletion and thymic atrophy, both cell types displaying active apoptosis.
The glucocorticoid-induced effects were similiar in type and level to those seen
in mice fed the zinc-deficient diet. These observations indicated that, in vivo,
increased glucocorticoid concentration but not reduced zinc concentration was
responsible for lymphopoaenia- and thymic atrophy-related apoptosis.
Other possible roles for zinc in apoptosis have been suggested to involve
inhibition of apoptotic nuclease activity, specifically that of DNAse 1 and the
endonuclease, NUC18 [164]. Both nucleases have been shown to be linked to
apoptotic DNA fragmentation and are inhibited by zinc ions.
Zinc uptake and cellular localization

Studies of cellular zinc uptake and subsequent localization within the cells have
traditionally utilized radiolabelled zinc although, more recently, novel fluoro-
metric methods have been developed [154-156]. These methods have allowed
103
the identification of both passive and active zinc uptake mechanisms [157,158].
Within the cell, zinc is mainly found bound to metalloproteins, with a small
unbound labile fraction in the cytoplasm. The metalloproteins include the
metalloenzymes, gene regulatory molecules and storage or carrier molecules
[2], which may be involved in the inflammatory process, examples of which are
mentioned below.
The matrix metalloproteinases are a class of metalloenzymes that require zinc
at the active site for catalytic activity [159]. The zinc-binding site is a conserved
histidine-glutamine-x-glycine-histidine sequence (where x is a hydrophobic
residue). Matrix metalloproteinases are responsible for much of the cartilage
destruction seen in osteo- and rheumatoid arthritis.
Metalloproteins such as metallothionein, rich in zinc-binding cysteine resi-
dues, act as storage and donating molecules for the zinc atom. However
metallothioneins have also been shown to have some involvement in inflamma-
tory states [160]. In the latter study, metallothionein was shown to have a
regulatory role in zinc uptake during LPS (lipopolysaccharide)-induced inflam-
mation in vivo where metallothionein and its concomitant zinc atoms helped to
maintain hepatic and blood glucose levels [160].
Free zinc has also been shown to have a synergistic action with stimulants of
the second messenger system involved in the production of monokines by
human peripheral blood mononuclear cells [161].
CONCLUSIONS
Zinc ions have an immense array of cellular and molecular actions manifesting
anti-inflammatory, immunoregulatory and antiulcer activities. Many of the
modes of its actions on these systems are incompletely understood. The
development of some zinc-containing compounds as drugs heralds a new focus
for treating a number of inflammatory conditions. The issue of the toxicity of
what appears, on the surface, to be a relatively non-toxic metal has yet to be
addressed, especially in the long-term application of novel zinc complexes for
therapy of some immunological and neurodegenerative diseases. Clearly, zinc
has profound effects on a number of physiopathological states and there is
considerable potential for manipulating the levels of this metal as a strategy for
controlling a variety of inflammatory and degenerative diseases.
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111
7
Copper complexes for therapy of cancer and
autoimmune diseases
JRJ Sorenson
Department of Medicinal Chemistry, College of Pharmacy,
University of Arkansas for Medical Sciences Campus, Little Rock,
Arkansas, USA
INTRODUCTION
This overview presents the pharmacological effectiveness of copper complexes
as anticancer and anti-inflammatory agents as a physiological approach to
treating cancers and autoimmune diseases [see References 1-8 and references
cited therein]. Plasma copper concentration increases in neoplastic and auto-
immune diseases as an immune-mediated physiological response to these
disease states. Treatment with copper complexes is a therapeutic support of this
increase in plasma copper and the attendant distribution of copper to affected
tissues to enable de-novo syntheis of copper-dependent enzymes required to
bring about remission by re-establishing normal tissue function.
Copper complexes also have antimutagenic, anticarcinogenic, and anti-
neoplastic activities which may be viewed as resulting from their facilitation of
the inflammatory responses required to overcome these disease states. The
antineoplastic activity is produced without cell killing.
Copper complexes to not inhibit any inflammatory response but produce
their anti-inflammatory effect by facilitating immune-mediated physiological
responses to overcome pathological events causing inflammation. Since all
diseases, and most particularly neoplastic diseases, have an inflammation
component, the antineoplastic activity of copper complexes can be viewed as
due to their antimutagenic, anticarcinogenic and anti-inflammatory activities. It
is concluded that anti-inflammatory agents that facilitate immune-mediated
physiological responses intended to overcome disease states may be effective in
treating neoplastic and autoimmune diseases.
113
K.D. Ralnsforri et a/. (eds.), Copper and Zinc in Inflammatory and Degenerative Diseases. 113-124.
CHANGES IN BLOOD COPPER CONCENTRATION ASSOCIATED WITH

NEOPLASTIC DISEASES [1,2]
There have been many reports of elevated plasma copper in neoplastic diseases.
Acute leukaemia is associated with an increase in serum or plasma copper and
bone marrow blast cells with the onset of active disease. These changes are
followed by a return to normal plasma copper with remission. Chronic
lymphatic and myeloid leukaemias as well as myelomas are associated with
decreased haematocrit, a possible symptom of copper deficiency, and near
normal copper levels that do not return to normal are always terminal. Active
Hodgkin's disease is always associated with elevated plasma copper which
returns to normal with remission and is diagnostic as well as prognostic.
Patients with non-Hodgkin's lymphoma also experience a return to normal
plasma copper with remission.
Patients with carcinomas also have elevated plasma copper levels. Progression
of cervical carcinoma through stages of increasing severity is associated with a
progressive increase in plasma copper with returns to normal with successful
therapy. Plasma copper is also elevated in mammary, bronchial, gastric, colonic,
rectal and liver carcinomas as well as osteosarcoma. The degree of serum
copper elevation in osteosarcoma has been correlated with the extent and
activity of this disease. Highest copper levels were associated with metastatic
disease and the poorest prognosis.
Irradiation-induced and spontaneous osteosarcomas remit following tumour
removal by limb amputation, with a return to normal plasma copper concen-
tration.
In each of these neoplastic disease states, elevated copper serum was found to
correlate with disease activity and returned to normal with remission. These
observations are consistent with the view that the increase in serum copper is a
physiological response required to facilitate remission.
The increase in blood copper concentration found in all neoplastic diseases
studied has special significance since many tumour cells have less than normal
copper-dependent and zinc-modulated superoxide dismutase (Cu-Zn-SOD)
activity in comparison with non-transformed cells. The elevation of blood
copper, in the form of ceruloplasmin (CP), copper albumin and copper amino
acid complexes secreted from liver parenchymal cells, may have a physiological
role in activating apoSOD and other copper-dependent enzymes in cancer cells
which playa role in facilitating remission and the subsequent return to normal
blood copper level when remission occurs.
CHANGES IN BLOOD COPPER CONCENTRATION ASSOCIATED WITH

IMMUNE-MEDIATED INFLAMMATORY DISEASES [3-5]
The increase in blood copper concentration found in arthritic disease was first
reported by Heilmeyer and Stuwe in 1938. Since then, there have been many
reports with marked elevations of 3--4 times the normal value in early active
114
COPPER COMPLEX THERAPY OF CANCER AND AUTOIMMUNE DISEASES
disease. Elevated plasma copper returns to normal with remission. Chronically

active disease is associated with only slight elevations, suggesting that there is an
inadequate response or inadequate copper intake associated with chronic diseae
and an inability to achieve the copper-dependent responses required to over-
come this disease state. The immune-mediated rise in blood ceruloplasmin,
amino acid-copper complexes, and the albumin--copper complex in response to
these inflammatory diseases may also be a physiological response directed
toward increasing distribution of copper to affected tissues and facilitating de-
novo synthesis of copper-dependent enzymes required to prevent further injury,
as with CuSOD synthesis, and facilitate repair of injury requiring other copper-
dependent enzymes, such as the lysyl oxidases and cytochrome C oxidase.
ANTICANCER ACTIVITIES OF COPPER COMPLEXES [1,2,4,5]

Sharples was the first to report that increasing the dietary copper of 4-
dimethylaminoazobenzene-fed rats lengthened hepatic tumour induction time.
Inorganic copper was also found to be effective in preventing ethionine-induced
liver tumours in rodents and a variety of other animal carcinomas.
However, treatment with inorganic copper was not as effective as therapy
with copper complexes. A single 5 mg/kg dose of Cu(II)(dimethylglyoximeh
increased life span of mice bearing Ehrlich ascites or Sarcoma 180 tumours to
2-3 times that of non-treated controls. Other copper complexes reported to have
similar antitumour activities in rodents are: Cu(II)(3,4,7,8,tetramethyl-l,1O-
phenanthrolineh 2+, Cu(II)(2-keto-3-ethoxybutyraldehyde-bis-thiosemi-
carbazone), Cu(II)(pyruvaldehyde-bis-thiosemicarbazone), copper complexes
of 2-formylpyridine and I-formylisoquinolone thiosemicarbazones, copper-
bleomycin, Cu(II)(glycyl-glycyl-histidinate), Cu(II)(glycyl-histidyl-lysine),
Cu(II)(pyridine-2-carboxaldehyde-2-pyridylhydrazonate), Cu(II)(salicyl-
aldehydebenzoylhydrazonate), Cu(II) amino acid complexes. Cu(II)(2,3,4-
trihydroxybenzaldoxyimateh, and a large variety of other classes of copper
complexes. In addition to demonstrating antitumour activity for these copper
complexes, mechanistic studies provided a great deal of information concerning
their possible inhibition of DNA synthesis, which may have been due to
redifferentiation of transformed cells.
Oberley and Buettner pointed out that all tumour cell lines have markedly
decreased superoxide dismutase activity. The marked decrease in manganese-
dependent SOD activity is consistent with a decrease in aerobic metabolism and
a decrease or only a modest increase in Cu-Zn-SOD activity is consistent with
an impaired response leading to transformation of differentiated cells to
neoplastic cells. As a result of the observed lowered SOD activities in tumour
cells and being aware of the SOD-mimetic activity of copper salicylate
complexes, Oberley and his students began to investigate the antitumour activity
of these and other copper complexes.
Oberley, Leuthauser, Buettner, Oberley, and Bize observed that copper-
dependent SOD obtained from porcine liver was effective in decreasing growth
115
of solid Sarcoma 180 tumours and prolonged the life of these tumour-bearing
mice. They also found that the copper complex of aspirin, Cu(IIh(acetyl-
salicylate)4, was more effective in decreasing tumour growth and prolonging life
than porcine-derived SOD. In order to generally increase the solubility and
specifically increase the lipophilicity of Cu(IIh(acetylsalicylate)4, which is
polymeric in the solid state, both dimethylsulphoxide and pyridine mono-
molecular solvates were prepared. Both of these solvates were found to be more
effective than the polymeric form. Since an increase in lipid solubility appeared
to enhance activity; an ether-soluble complex, Cu(IIh(3,S-DIPS)4' was also
studied and found to be the most effective salicylate complex studied.
In addition to being effective against Sarcoma 180 tumours, Cu(IIh(3,S-
DIPS)4 as well as Cu(IIh(salicylate)4 and Cu(IIh(3,S-ditertiarybutylsalicylate)4
also inhibited growth of solid Ehrlich carcinomas and markedly increased the
life span of mice bearing this solid tumour. Increasing the number of treatments
further increased host survival time by inhibiting metastasis and decreasing
tumour growth.
Treatment with Cu(IIh(3,S-DIPS)4 was also found to have an additive effect
on survival of solid Ehrlich tumour-bearing mice when it was co-administered
with the anticancer agent 1,3-bis(2-chloroethyl-2-nitrosourea) (BCNU). A
single dose of 14.S mg/kg Cu(IIh(3,S-DIPS)4 was essentially ineffective in
increasing survival and a 30 mg/kg dose of BCNU produced only IS% survival
while the combination of these two agents produced a 60% survival through the
course of the experiment. These results were interpreted as being consistent with
the SOD-mimetic activity of Cu(IIh(3,S-DIPSk
Superoxide dismutase mimetic activity ofCu(IIh(3,S-DIPS)4 also led to studies
of this compound's ability to inhibit interleukin-2 (IL-2) synthesis by a mouse
thyoma cell line, EL4. Cu(IIh(3,S-DIPS)4 did inhibit phorbol diester-induced
synthesis ofIL-2 (IC5o =10 IlmollL) but it did not inhibit phorbol diester-induced
attachment ofthese cells to substrate. While 3,S-DIPS also inhibited IL-2 synthesis
(IC 50 =IS IlmollL), a 100 IlmollL solution of Cu(II)Ch was ineffective. Mechan-
istically, the inhibition ofIL-2 synthesis by Cu(IIh(3,S-DIPS)4 was suggested to be
due to an inhibition ofIL-2 messenger RNA transcription.
Leuthauser also observed that solid Ehrlich tumours taken from mice treated
with Cu(IIh(3,S-DIPS)4 contained differentiated epithelial cells in duct
arrangement, suggesting that Cu(IIh(3,S-DIPS)4 treatment did not kill these
mammary-derived tumour cells but caused them to differentiate to normal duct
cells. This observation was confirmed and extended by Sahu in his dissertation.
He found that Cu(IIh(3,S-DIPS)4 added to neuroblastoma culture medium
caused differentiation of these neoplastic cells to normal neuronal cells in a
concentration-related manner. If Cu(I1h(3,S-DIPS)4 does not kill transformed
cells but instead causes them to differentiate to normal cells, the future use of
copper complexes to treat neoplastic diseases has some exciting possibilities.
Recent studies of Cu(I1h(3,S-DIPS)4 have demonstrated that this binuclear
complex is in equilibrium with its mononuclear complex, Cu(II)(3,S-DIPS)4' in
polar solutions. Electron spin resonance studies revealed that the mononuclear
116
complex interacts with Ehrlich cell membranes and appears to be rapidly

absorbed with less impedance due to strong lipophilic bonding interactions with
cell membrane macromolecules. Solubilization in these cell membranes would
account for rapid absorption observed following interaction of Cu(IIh(3,5-
DIPS)4 and Cu(II)(nirvanolh(pyh with components of the Ehrlich cell mem-
brane and, perhaps, subsequent reduction by GSH or some other thiol-contain-
ing cellular component.
In 67 Cu pulse-chase experiments using hepatoma cells, Freedman and Peisach
found that 67Cu-Iabelled GSH was reversibly formed in an exchange sequence
with [67 Cu]thionein. 67Cu-Iabelled CU2Zn2S0D was presumed to be the result of
an exchange reaction between [67 CU]GSH and apoenzyme. Transfer of Cu(I)
from MT to GSH and then to Zn2S0D, which was subsequently supported by
Ciriolo et al. using ESR, NMR and optical spectrophotometry, suggests a role
for GSH in mediating transfer of Cu(I) from Cu(I)thionein to Zn2S0D.
However, other Cu complexes may also serve in direct transfer to the Zn-
containing apoenzyme.
New Cu complexes have been found to have anticancer activity. A very stable
Cu(II) complex of daunorubicin, which does not stimulate oxyl-radical
production as daunorubicin does, was suggested to be a promising approach to
treating neoplastic diseases without producing cardiotoxic effects. Other SOD-
mimetics, Cu(II)[1 ,8-di(2-imidazoyl)-2, 7 -diazocladiene-l, 7](CI0 4h and
Cu(II)[N,N-bis(2-pyridylmethylene)-1 ,4-butanediamine](CI04h, both charged
complexes, were found to be cytotoxic to human K562 erthroleukaemia cells.
The mechanism of action was suggested to involve GSH oxidation and H 20 2
formation rather than exclusive disproportionation of 02". Low GSH
peroxidase activity in these neoplastic cells, compared with normal human
lymphocytes, coupled with a depletion of GSH and increased production of
H 20 2 was suggested to account for greater efficacy of these complexes in
eliminating these neoplastic cells at concentrations that did not affect human
lymphocytes. It is most likely that re-establishment of Cu-dependent enzymes
and proteins are the principal mechanistic parameters accounting for anticancer
activities of Cu complexes. All these data support the use of Cu complexes as a
physiological approach to preventing and treating human cancers.
ANTI·INFLAMMATORYACTIVITIES OF COPPER COMPLEXES [1,2,4,5]

Copper complexes have been shown to be effective anti-inflammatory agents in
all animal models of inflammation following oral or parenteral administration.
They are always more effective than the parent ligand, whether or not the parent
ligand has anti-inflammatory activity. If the parent ligand has anti-
inflammatory activity, the copper complex is 5 to 30 times more effective than
the parent ligand. Active doses are non-toxic and provide amounts of copper in
the range of recommended daily intake of copper. In addition, these copper
complexes lack the principal toxicity of the parent anti-inflammatory drugs,
ulcerogenicity, and they are potent anti-ulcer agents.
117
SOD-MIMETIC ACTIVITY OF COPPER CHELATES [1,2,4,5]

Following the original report that copper chelates of antiarthritic drugs are
more active and less toxic anti-inflammatory agents than their parent drugs, De
Alvare et al. and many others suggested that 02" disproportionation, via a ping-
pong mechanism, accounted for this activity. This continues to be the subject of
many reports addressing mechanistic aspects of anti-inflammatory and anti-
ulcer copper chelates including: Cu(IIh(salicylate)4 and Cu(IIh(indo-
methacinate)4, pyridine solvates of Cu(IIh(aspirinate)4' substituted pyridine
solvates of tetrachlorocuprate(II), Cu(IIh(salsalate)4, Cu(II)-[aminomethyl-
4(1,I-dimethylethyl)-6-iodophenol], histidine analogues, Cu(II)N03 and
Cu(II)(Glyh, and Cu(II)(L-Trp)(L-Phe), and Cu(II)S04, Cu(II)(6-benzyl-
arninopurineh, Cu(II)(kinetinh, Cu(IIh(3,5-DIPS)4' Cu(I)(2,9-dimethyl-l, 10-
phenanthrolineh(N0 3), Cu(II)(2,9-dimethyl-l, 10-phenanthrolineh(N03h,
Cu(II)(5-methyl-l-l'-bipyridineh(N0 3h, Cu(II)(2,9-disubstituted-l, 10-
phenanthrolines)(N0 3h, Cu(II)( l-substituted-3-(1-pyridine)-isoquinolonesh
(N0 3h, Cu(I1)(N-salicylidene-aminoalcanoate),L - and D ,L K[Cu(I1)(N-
salicylide-glutamato)(H20)], K[Cu(II)(isothiocyanato)-N-salicylideneglycinato],
K[Cu(II)(isothiocyana to )-N-salicylidene- ~-analina to], K[Cu(II)(iso-
thiocyanato)-N-salicylideneglycinato]; Cu(II)(aspirinateh(DMSOh, Cu(II)(as-
pirinateh(imHh, Cu(II)(aspirinateh( 4-pich, Cu(II)(aspirin ate h( 1-Meimh,
Cu(II)(aspirinateh(pyh, Cu(II)(salicylateh, Cu(II)(Gly-Ala)(bipy), Cu(II)(Gly-
Ala)(phenanthroline), Cu(II)(Gly-Phe)(bipy), Cu(II)(Gly-Phe)(phenanthroline),
Cu(II)(Gly-Tyr»(bipy», Cu(II)(Gly-Tyr)(phenanthroline), Cu(II)(Gly-
Tyr(dmph), Cu(II)(Gly-Gly)(bipy), Cu(II)(Gly-Gly)(phen), and Cu(II)(Gly-
Gly«dmph), Cu(II)(ranitidineh, Cu(II)(cimetidineh, Cu(II) (histamineh and
Cu(II)(Hish, Copper chelates of still newer anti-inflammatory agents, pirox-
icam and tenoxicam, have been synthesized and characterized; however, no
SOD-mimetic, anti-inflammatory or antiulcer data have as yet been reported for
them.
Contrary to the suggestion that it is unlikely that small-molecular-mass
copper chelates, such as Cu(IIh(3,5-DIPS)4 disproportionate O2 in blood
plasma, Huber et al. reported that many such complexes, including
Cu(IIh(3,5-DIPS)4 are more effective in removing 02" than bovine RBC
CU2Zn2S0D in fetal calf serum and found that ternary Cu(IIh(3,5-DIPS)4-
albumin complexes do disproportionate 02".
While it is likely that copper chelates undergo ligand exchange in vivo, it is
this exchange that is required for cellular utilization and must account for the
activation of inactive apoenzymes and the beneficial pharmacological effects of
copper chelates. Also, it is well known that most administered drugs do not
reach the site of disease pathology. Up to 99.999% of administered drugs may be
removed by the liver and/or be distributed to tissues other than the disease-
affected tissue(s). Consequently, not all of an administered copper chelate is
expected to reach the target tissue. The effective anti-inflammatory dose of
Cu(IIh(aspirinate)4 is 1 flmollkg or 70 flmol for an 'average' adult and
118
represents 10 19 molecules/adult. If only 0.001% of this dose was distributed

throughout the body, which is composed of an estimated 10 12 cells, there would
be 100 molecules of drug for every cell in the adult body. Since there is always
vasodilation at the site of disease-affected cells, as a component of the
inflammatory response, and greater perfusion of the affected cells by plasma
filtrate containing the drug, the concentration of drug in the environment of
disease-affected cells would actually be far greater than 100 molecules/cell.
DOWN·REGULATION OF MONO·OXYGENASES [5]

Earlier reported modulation of aminopyrine-N-demethylase and aniline p-
hydroxylase activities by SOD-mimetic copper chelates continues to be
mechanistically interesting. Recent determinations of 25% inhibitory concen-
trations (10 25 ) of Cu(II) sulphate, 47 and 10.5 JlIllollL respectively in these two
P450 systems, were found to be smaller than 1025 concentrations of Zn(II)
sulphate, 226 and 330 JlmollL respectively. It is unclear whether these high
concentrations of zinc gave chelates which were specific for the loss of enzyme
activity.
NEW ANTI·INFLAMMATORY COPPER CHELATES [6]

An earlier review [5] lists over 140 copper chelates; all are more active as anti-
inflammatory agents than their parent compounds. Similar comparisons of the
effective molecular mass of parent ligands with the effective molecular mass of
their chelates, which is most appropriate when comparing pharmacological
effects of compounds have markedly different molecular masses, physico-
chemical properties, and given subcutaneously (sc), orally or topically, continue
to provide additional evidence for increased activity of the copper complexes.
Copper(IIMaspirinate)4 given as oral doses of 0.24 and 0.47 mmollkg produced
16 or 72% inhibition, with altered tissue Cu, Zn and Fe contents. Acute
inflammation in this model was also dramatically reduced with oral adminis-
tration of 0.12, 0.24 or 0.47 mmollkg Cu(IIMaspirinate)4 producing 30, 38 or
54% inhibition of paw inflammation while larger doses of 0.56, 1.11. and 2.22
mmollkg aspirin produced only 8, 31 or 54% inhibition. Acute carrageenan paw
oedema (CPO) was also dramatically reduced following oral administration of
Cu(IIMaspirinatek A 0.1 mmollkg dose produced 56% inhibition while a 0.6
mmollkg dose of aspirin produced only 33% inhibition.
Copper chelates of salicylidine-anthranilic acid Schiff bases were found to be
orally effective and more active as anti-inflammatory agents in rat CPO and as
antiulcer and antidiarrhoeal agents than their parent drugs. Substituted bis-
pyridino solvates of tetrachlorocuprate che1ates were found to be more effective
than indomethacin, phenylbutazone or triamcinolone in CPO, CWG, croton
oil, or PA models of inflammation. Dimethylsulphoxide-glycerol solvates of
Cu(II)(pheny1butazone)z and Cu(II)(sa1icylate)4 che1ates were more effective
anti-inflammatory agents than their parent compounds in rat CPO and in the
119
COPPER AND ZINC IN INFLAMMATORY AND DEGENERAllVE DISEASES
carrageenan alar-fold oedema horse model of inflammation when applied

topically. Copper(IIh(gluconate)4, gold thiomalate, a silver proteinate, and
Cu(IIh(flurbiprofen)4 were also more active than their parent compounds in
various animal models of inflammation, pain and gastrointestinal injury and
they caused less gastric irritation.
The early observation that mixtures of solutions of inorganic copper and non-
steroidal anti-inflammatory drugs are less effective than synthesized chelates has
been confirmed by Korolkiewicz et al. by comparing mixtures of CuN0 3 and
salicylic acid or aminopyrine with copper chelates of these two drugs and by
Milanino et al. with a series of 2-benzimidazoylthioether copper chelates in
CPO and PA following oral administration. Milanino et al. also reported that
indomethacin is significantly less effective in copper-deficient rats with CPO.
BIODISTRIBUTION OF COPPER IN INFLAMMATION [5,6]

Biodistribution studies using 64Cu(IIh(salicylate)4 and Nas 64CU(I)8 64Cu(IIMD-
penicillamine)I2CI in several models of inflammation following topical or
subcutaneous administration revealed that 64copper is sequestered at the site of
inflammation. These results support the hypothesis that applied copper chelates
lead to the appearance of copper in target tissues and offer an explanation as to
how applied copper chelates can be anti-inflammatory. However, 64copper in
the D-pen chelate was rapidly excreted via urine by normal mice and rats as well
as rats with inflammation. Doubled labelled 67Cu(IIh[3,5-DIP-(carboxy-14C)S]4
was found to rapidly distribute to blood, liver, intestine, femur, spleen, thymus,
kidney, lung and brain within 0.5 h of administration and underwent slow
ligand exchange wherein 3,5-DIPS slowly disappeared from blood, intestine,
spleen, thymus, kidney, lung and brain over a 24-h period. The radioactive
copper persisted in these tissues for the 5-day duration of the study. Both ligand
and copper were found in liver, femur and muscle throughout the entire 5-day
period of study. These data support the hypothesis that Cu(IIh(3,5-DIPS)4 is a
bioavailable form of copper and that ligand exchange does occur with
utilization of administered copper.
FORMATION OF COPPER CHELATES IN VIVO [5-7]

D-Pen, gold(I)thioglucose, and gold(I)thiomalate which are used to treat
rheumatoid arthritis were suggested to remove copper bonded to albumin or
Cu thioneine by ligand exchange to account for their antirheumatic activity and,
although the existence of a D-Pen disulphide chelate in vivo has been
questioned, its use as a drug may still offer effective antirheumatic therapy. The
copper chelate of thoimalate is also more effective as an anti-inflammatory
agent than gold thiomalate. Treatment of arthritics with a modest dose of
Cu(II)S04 plus D-Pen produced no clinically important results. More
appropriate treatment with a less-polar form of copper, Cu(II)(Glyh, but still
a modest dose containing only 2 mg copper, for only 4 weeks, did increase
120
erythrocyte CU2Zn2S0D activity in 18 of 23 arthritic patients, consistent with

pre-existing marginal copper status in these RA patients. A recently described
CP receptor on human erythrocytes offers promise with regard to identification
of CP receptors on other cell types and a better understanding of the role of CP
in overcoming arthritides.
Only mixtures of D-Pen, glutathione (GSH) or other thiols with copper
significantly depressed in-vitro DNA synthesis in proliferating human endo-
thelial cells and rabbit corneal neovascularization in a dose-dependent manner
as measures of plausible antiangiogenic activity in treating rheumatoid arthritic
disease. Neither thiol nor Cu sulphate alone affected DNA synthesis. These
results were interpreted to indicate that thiol copper chelates produce a
pronounced antiangiogenic effect in inhibiting endothelial cell proliferation. It
was suggested that rheumatoid synovitis was suppressed by reducing the
number of small blood vessels available for emigration of chronic inflammatory
cells (mononuclear and polynuclear lymphocytes) and the consequent pro-
liferation of synovial tissue. These copper chelates may have actually had a role
in tissue repair, facilitating collagen and elastin cross-linking, as well as enabling
subsidence of other inflammatory responses.
INHIBITION OF HAEMOLYSIS, FEVER AND SPLEEN CELL ANTIBODY

SYNTHESIS AND PROLIFERATION [7]
A Cu-dextran complex plus aspirin reduced lypopolysaccharide (LPS)-induced
RBC hypotonic haemolysis for up to 6 h and fever for up to 4 h in rabbits, while
neither drug given alone had any affect on body temperature. Singlet O 2-
induced haemolysis has been prevented by Cu(II)(Glyh, Cu(II)(Tryh, and
Cu(II)(cimetidineh and Cu(II)(Glyh failed to cause haemolysis in vivo.
Yamauchi and Yamamoto found that Cu(II)S04 suppressed spleen cell anti-
body synthesis and pokeweed and LPS mitogenicities as an inhibitor of B-cell
proliferation.
MODULATION OF PMNL ACTIVITIES [4-7]

It has been suggested that O2 induces de-novo synthesis of interleukin-l (IL-l) or
an IL-l-mimetic agent by polymorphonuclear leucocytes (PMNLs) is a model of
up-regulation of the PMNL-mediated inflammatory response which may be due
to a translation-mediated up-regulation of protein kinase C. In a preliminary
study, it was demonstrated that 0.5 mmol/L Cu(IIh(aspirate)4 was more effective
than 0.5 mmollL aspirin in inhibiting N-formyl-methionyl-Ieucyl-phenylalanine
(fMLP)-induced chemokinesis and chemotaxis although the inhibition of
chemotaxis by Cu(I1h(aspirinate)4 was greater than the inhibition of chemokin-
esis. Rat PMNLs obtained following induction of isologus serum pleurisy and
treatment with 0.6 mmollkg Cu(I1h(aspirinate)4 showed reduced fMLP-induced
chemotaxis with no effect on chemokinesis while treatment with 2.8 mmollkg
aspirin produced no effect on either chemokinesis or chemotaxis. In a subsequent
121
comparison of aspirin, 3,5-DIPS, and indomethacin with Cu(IIh(aspirinate)4'

Cu(IIh(3,5-DIPS)4 and Cu(IIh(indomethacinh to determine the minimum
effective concentration of these compounds, it was found that 8.3 mmol/L aspirin
and 0.2 mmollL Cu(IIh(aspirinate)4 decreased both chemokinesis and chemo-
taxis; 1.1 mmollL 3,5-DIPS and 0.05 mmol/L Cu(IIh(3,5-DIPS)4 were equi-
potent but only decreased chemotaxis; and 28 IlmollL indomethacin and 61lIDol/
L Cu(IIh(indomethacin)4 were also equipotent but only decreased chemotaxis.
The large but smallest effective concentration of Cu(IIh(acetate)4, 250 llIDollL,
decreased both chemokinesis and chemotaxis. Copper(IIh(aspirinate)4'
Cu(IIh(3,5-DIPS)4 and Cu(IIh(indomethacinate)4 were also more effective in
decreasing calcium pyrophosphate-induced pleural exudate and the number of
PMNLs in these exudates than their parent ligands as well as Cu(IIh(acetatek
The order of in-vivo anti-inflammatory activity for these chelates was:
Cu(IIh(indomethacinate)4 > Cu(IIh(3,5-DIPS)4 > Cu(IIh(aspirinatek
Copper(IIh(acetate)4 was either ineffective or exacerbated pleuritic inflamma-
tion.
Down-regulation of phorbol diester-stimulated PMNL O 2 production by
Cu(I1h(3,5-DIPS)4 and CU(I1)S04 may be primarily due to modulation of
protein kinase C. This observation offers a clearer understanding of reports that
PMNLs have reduced O 2 uptake and produce less O 2 when exposed to
Cu(I1)(salicylateh, Cu(I1h(3,5-DIPS)4 or Cu(IIh(indomethacinate)4 and why
PMNLs isolated from calcium pyrophosphate-induced pleural exudates
exhibited decreased chemotaxis following treatment with Cu(I1h(aspirinate)4,
Cu(IIh(3,5-DIPS)4 or Cu(IIh(indomethacinatek Copper(I1)(3,5-DIPS)4 and
Cu(I1h(indomethacinate)4 were also shown to have SOD-mimetic activity in the
phorbol-stimulated granulocyte respiratory burst system. Results reported by
Nilsson also provide mechanistic understanding to account for the modulation
of chemokinesis and chemiluminescence in Cu(I1h(aspirinatek, Cu(IIh(3,5-
DIPS)4- and Cu(IIh(indomethacinate)4-treated cultures of PMNLs or in vivo
following calcium pyrophosphate-induced pleurisy. Down-regulation of
PMNLs by CP, however, must be due to extracellular removal of O 2. This
modulation of O 2 production by copper chelates does not compromise PMNL
function since Cassone has shown that phagocytosed bacteria have a shorter
residence time in PMNLs treated with Cu(I1h(aspirinate)4 than those treated
with aspirin, and copper has myeloperoxidase-mimetic activity unlike the
inhibition of myeloperoxidase by many anti-inflammatory drugs. The
modulation of protein kinase C activity by copper chelates is also consistent
with the possibility that Cu(II)(salicylateh prevented the reduction of spin-
labelled cell membranes by O 2 following phorbol ester activation of PMNLs or
reacted with it such that the spin label was within the cell membrane. The
antimicrobial activity of copper chelates may also support PMNL function.
Additional support for the use of copper chelates in supporting PMNL function
comes from observations by Babu and Failla that copper deficiency reversibly
depresses neutrophil function in terms of peritoneal macrophage O 2 production
and candidacidal activity.
122
MODULATION OF EICOSANOID SYNTHESES [1,2,4-7]

Conversion of macrophage labelled [14C]arachidonate to various eicosanoids,
prostaglandin E2 and thromboxane2 A2, via stimulation of phospholipase A2
activity was decreased in a time-dependent fashion with medium containing 5-
10 ~mo1/L Cu(IJ) sulphate or Cu(IJ) nitrate but markedly increased these
eicosanoids along with prostaglandin F 2a with 50-100 ~mo1/L Cu(IJ) sulphate
without increasing trypan blue uptake. There was no additive effect with
exogenous addition of copper and arachidonate. The addition of 100 ~01/1
Cu(II) sulphate also stimulated lysosomal secretion of ~-glucuronidase.
Copper(IJ) nitrate, but not zinc(IJ) sulphate, also stimulated eicosanoid
syntheses and enzyme secretion. These results were interpreted as consistent
with a copper-dependent stimulation of phospholipase A2 activity. There were
also increases in HETE, TXB2 and PGF2a and decreases in PGA2 and PGE2
which are consistent with the down-regulation and modulation of the
inflammatory response, found when accounting for the total recovered 14C
radioactivity. The large concentrations of Cu(IJ) sulphate and Cu(IJ) nitrate
required for these observations suggest that the use of copper chelates to achieve
these effects may allow the production of these effects with more relevant
physiological concentrations of copper. Copper chelates of substituted 1,2,3-
triazole-2-propionic acids were also found to stimulate prostaglandin syntheses
while their parent ligands were either less stimulatory or inhibited cyclo-
oxygenase as anticipated. Modulation of gastric eicosanoid syntheses has also
been suggested to account for promotion of mucus effusion by Cu(II)bis-2-
benzimidazoylthioethers. Consistent with the role of copper chelates in
modulating the inflammatory response, Cu(IIh(aspirinate)4 and Cu(IIh(indo-
methacinate)4 were reported to decrease phospholipase A2 activity.
SUMMARY OF EARLIER REPORTED BIOCHEMICAL EFFECTS OF

COPPER CHELATES [1,2,4-7]
In addition to the above recent mechanistic studies, Cu complexes have
previously been reported to modulate: lysyl oxidases, eicosanoid syntheses and
arachidonate metabolism, CU2Zn2S0Ds and demonstrate SOD-mimetic-
activity, stabilization of synovial and polymorphonuclear leucocyte lysosomal
membranes, histamine activity, lymphocyte mitogenic and chemotactic
responses, stabilization of 'Y-globulin, GSH status, GSH-S-transferase, pre-
prothrombin carboxylase, cytochrome P450 mono-oxygenases, and/or
NADPH-dependent P450 reductases, 2-oxoglutarate-dependent hydroxylase,
lipid peroxidation, angiogenesis and luteinizing hormone-releasing hormone
secretion. Further support for the use of copper chelates in the treatment of
inflammatory diseases comes from the observation that Cu(IIh(3,5-DIPS)4
down-regulates nitric oxide synthase [8].
123
TREATMENT OF INFLAMMATORY DISEASE IN MAN [1,4,5]

Copper chelates have been successfully used to treat rheumatoid and other
chronic degenerative diseases. Possible alternative structures of one of these,
cupralene, have recently been suggested to be 1: 1 and 1:4 complexes. Patents
have recently been issued for the treatment of acne with copper chelates of
lanolin fatty acids and for the use of copper chelates as antiarthritic and anti-
inflammatory drugs with antiulcer activity.
All of the above support the use of copper chelates or complexes as a
physiological approach to treating arthritic and other inflammatory diseases of
man, which is a very exciting possibility despite offerings of uninformed
ambivalent opinion.
REFERENCES
1. Sorenson JRJ, cd. Inflammatory Diseases and Copper. Totowa, NJ: Humana Press; 1982.
2. Sorenson JRJ, ed. Biology of Copper Complexes. Totowa, NJ: Humana Press; 1987.
3. Sorenson JRJ. An evaluation of altered essential metal concentrations in rheumatoid
arthritis. Inorg Perspec Bioi Med. 1978;2:1-26.
4. Sorenson JRJ. Copper complexes in the treatment of experimental inflammatory conditions:
inflammation, ulcers, and pain. In: Rainsford KD, Velo GP, eds. Copper and Zinc in
Inflammation, Volume IV. Lancaster, UK: MTP Press; 1989: 69-84.
5. Sorenson JRJ. Copper complexes offer a physiologic approach to treatment of chronic
diseases. Prog Med Chern. 1989;26:437-568.
6. Sorenson JRJ. Copper-potentiation of nonsteroidal antiinflammatory drugs. In: Berthon G,
ed. Handbook of Metal-Ligand Interactions in Biological Fluids: Bioinorganic Medicine,
Volume 2. New York: Marcel Dekker; 1995: 1318-24.
7. Sorenson JRJ. Pharmacological activities of copper compounds. In: Berthon G, ed. Hand-
book of Metal-Ligand Interactions in Biological Fluids: Bioinorganic Medicine, Volume 2.
New York: Marcel Dekker; 1995: 1128-39.
8. Baquial JGL, Sorenson JRJ. Down-regulation of NADPH-diaphorase (nitric oxide synthase)
may account for the pharmacological activities of Cu(U)z(3,5-diisopropylsalicylatek J Inorg
Biochem. 1995;60: 133-48.
124
8
Zinc and copper in the treatment of
rheumatic diseases
F Fernandez-Madrid
Division of Rheumatology, Department of Internal Medicine, Wayne
State University, Hutzel Hospital. 4707 St Antoine Boulevard,
Detroit, MI 48201, USA
ZINC
Zinc has an important role in human health because of its participation in
multiple enzymatic reactions and its involvement in the immune system [1,2].
Relevant to the treatment of the rheumatic diseases, zinc deficiency profoundly
disturbs the immune function and the host defense mechanism, resulting in
lymphoid atrophy, impaired delayed hypersensitivity and T-helper cell dysfunc-
tion [2-5].
Zinc administration can reverse the cellular immunodeficiency observed in
patients with marasmus and kwashiokor prior to calorie or protein replenish-
ment [6]. Zinc deficiency can occur relatively quickly under certain circum-
stances because there are no major body storage depots of zinc which are readily
available. Zinc deficiency has been observed in patients with chronic diseases,
such as malabsorption syndromes, chronic renal failure, cirrhosis of the liver,
sickle cell disease, acrodermatitis enteropathica and other chronic debilitating
diseases [1,2].
Zinc status has been actively investigated in patients with rheumatic diseases,
particularly in rheumatoid arthritis (RA) because of their chronicity.
Low serum Zn and high serum eu are characteristic of RA [7-14]. In
multiple linear regression models, both can be explained by disease activity
parameters [11]. Milanino et al. have thoroughly studied copper and zinc status
in 120 patients with rheumatoid arthritis [13]. As had previously been reported,
they found that plasma zinc levels were significantly lower in patients with RA
than in controls. However, these authors reported that, in terms of mean
erythrocyte corpuscular content, RA patients and control subjects possessed
equal amounts of zinc in their blood cell fraction [13]. Therefore, Milanino et al.
proposed that the RA group of subjects examined was not deficient in zinc [13].
This result appears solid for the whole group of 120 patients but the analyses for
differences among groups according to disease duration or anatomical class by
125
K.D. Rainsford et a/. (eds.), Copper and Zinc in Inflammatory and Degenerative Diseases. 125--137.
necessity considered a limited number of patients. Milanino et al. also showed

that plasma zinc concentrations in patients with RA correlated not only with
acute-phase reactants, as did eu, but also with haemoglobin concentration and
with all clinical parameters, including swollen joints, grip strength, functional
and anatomical stage and physician assessment [13]. These authors, as well as
Mussalo-Rauhamaa et al. [15] have suggested that plasma zinc concentration
could have some practical value in defining the overall severity of the disease.
The data of Milanino et al. show that the use of steroids and/or NSAIDs is
associated with significantly lower plasma levels of zinc than those measured in
patients not taking these drugs. However, patients taking NSAIDs or steroids
frequently have more severe disease than those not requiring these therapies.
Previous studies have also noted that the therapy received by patients with RA
may influence plasma zinc levels [14].
The potential therapeutic value of zinc in the rheumatic diseases has been
previously reviewed [16]. The initial reports oflow plasma zinc in patients with
RA were thought to represent zinc deficiency and led to the use of oral zinc
supplementation in the treatment of RA [17]. Simkin's report [17] elicited great
interest and triggered further investigations into the relationship between zinc
and RA. Few investigations have focused on the dietery trace metal intake of
patients with RA [18-20]. These studies have consistently found a deficient
intake of zinc and copper in patients with RA. Although low plasma zinc levels
have been reported in numerous studies in patients with rheumatoid arthritis,
the extent to which deficient intake contributes to these low serum zinc levels is
unknown. Peretz et al. have assessed the phagocytic ability of blood polymor-
phonuclear cells (PMNs) by cytofluorometry in a group of patients with
inflammatory rheumatic diseases before and after oral zinc supplementation
[21]. Phagocytosis was reported to be significantly impaired in patients with
rheumatic diseases and these changes could be corrected by zinc supplementa-
tion. The clinical significance of this effect is unknown but deserves further
investigation. Peretz et al. also investigated the effects of zinc supplementation
on zinc status and on clinical and biological indicators of inflammation in a
group of 18 patients with chronic inflammatory rheumatic diseases and in 9
healthy control SUbjects. No differences in clinical or biological parameters were
observed between these patients supplemented with 45 mg Zn/day (as gluta-
mate) or placebo during the 60-day trial. The results were inconclusive because
of the small number of subjects and the relatively short duration of the
experiment [22].
Honkanen et al. suggested that the effect of interleukin-l (IL-l) may be
responsible for the low serum zinc and high serum eu characteristically found
in RA [11]. This cytokine would increase the metallothionein-mediated hepatic
uptake of zinc and would upregulate ceruloplasmin gene and increase its
synthesis in the liver, resulting in higher levels of ceruloplasmin-eu complexes
in the blood. It has been reported that IL-l and IL-6 are significantly increased
in plasma of patients with RA [23]. IL-l, probably acting through IL-6
production [24], has been shown to promote a redistribution of zinc, inducing,
126
METALS IN RHEUMATIC DISEASES
in particular, the accumulation of the metal in the liver and its concomitant
decrease in plasma [25]. It is possible that these findings may explain the
hypozincaemia found consistently in patients with RA [11,13]. Although it is
clear that the majority of the patients studied by Milanino et al. were not zinc
deficient [13], marginal deficiences of zinc are likely to be present in some
subsets of RA, such as those with stage III-IV disease as well as in chronically
ill patients treated with NSAIDs and corticosteroids.
Although the beneficial effect of oral zinc supplementation initially reported
in patients with RA [17] could not be confirmed in subsequent studies [26-28],
experiments on animal models of rheumatoid arthritis have shown an anti-
inflammatory effect of zinc administrationm [29,30]. Moreover, recently Naveh
et al. [31] investigated zinc absorption in patients with RA using a zinc-tolerance
test and urinary excretion according to the method of Sullivan et al. [32]. They
found that, upon ingestion of 50 mg elemental zinc, plasma zinc in a group of
healthy volunteers rose from 111 ± 7 Ilg/dl to a peak of 200 ± 24 Ilg/dl (mean ±
SEM) in 2 h, while, in groups of rheumatoid arthritis patients with low and high
disease activity, the level of plasma zinc did not change significantly after zinc
ingestion. As reported in previous studies, plasma zinc before ingestion was
significantly lower in the rheumatoid patients than in the control group. Twenty-
four-hour urinary zinc excretion before and after zinc ingestion was significantly
lower in the group of patients with RA than in the control group. These authors
speculated that low plasma zinc widely reported in patients with RA may be the
consequence of zinc malabsorption and may result in zinc deficiency [31]. Little
is known about the homeostatic mechanisms that regulate the amounts of zinc
that are absorbed by the gastrointestinal tract [2,33] and clearly more research is
needed to elucidate this physiological event under normal conditions as well as
in rheumatoid arthritis. Zinc is absorbed in the jejunum and ileum and this
process is inhibited by phytic acid, calcium and other compounds [33]. In
consequence, zinc absorption studies in rheumatoid arthritis should ideally use
a diet rigorously controlling for those factors which may retard zinc absorption.
It is evident that the abundant literature in favour of or against an effect of
zinc supplementation in the treatment of RA does not settle this issue. The
different results obtained in various studies appear to be explained by the
enormous heterogeneity of the population of patients with rheumatoid arthritis.
The methodological problems encountered in the design of these studies appear
almost insurmountable given the chronicity of the disease, the differences in
clinical settings as well of functional and anatomic status, socioeconomic
conditions, dietary restrictions and multiplicity of drugs used in the therapy of
this disease. Although patients with a high socioeconomic level and adequate
nutrition are not likely to be zinc deficient, patients in lower socioeconomic
levels, those on aberrant or deficient diets and those treated with corticosteroids
are likely to have marginal zinc deficiency. Modest zinc supplementation in the
presence of these risk factors is favoured by the lack of toxicity of zinc
supplementation in moderate doses. In spite of the remarkable lack of toxicity
of ingestion of this trace element in humans [34], the common food fad of
127
unsupervised zinc supplementation in great excess of the recommended dietary

allowance is to be discouraged [35].
Zinc status and copper status have been investigated in other rheumatic
diseases. The distribution of Cu and Zn was determined in sera from patients
with the complete type of Behcet's disease in the active stage and in sera from
normal healthy controls [36]. These authors found that the concentrations of
Zn, as well as those of Se and Rb, were significantly lower for patients than
control subjects. However, there was a significant increase in Cu so that the Cui
Zn ratios were significantly higher for patients than for control subjects. An
increased Cu concentration had been previously reported in the sera from
patients with Behcet's disease [36]. These changes are not unlike those seen in
other chronic diseases and are probably non-specific, secondary to chronic
inflammation.
Although outside the scope of this review, it should be mentioned that zinc
may be involved in the process of cartilage degradation in the rheumatoid joint
which has been attributed to matrix metalloproteases [37-39]. Matrix metallo-
proteinases (MMP) are zinc metalloenzymes [2] and, on this basis, zinc has been
implicated in the pathogenesis of RA. The metal is bound firmly to MMPs and
participates directly in enzyme catalysis [2]. While there is great interest in the
potential therapeutic effect of MMP inhibitors, very little is known about the
effect of changes in zinc status on the activity of these enzymes in affected joint
tissues.
At the present time, we still face several questions related to zinc status in the
rheumatic diseases. Current evidence suggests that dietary intake of zinc and
copper is frequently deficient in patients with RA [18-20]. It has been suggested
that with a 'replacement' containing about 100% of the RDA for these trace
metals, most patients would do well [20]. However, if zinc absorption is
defective in patients with RA as suggested in the interesting report by Naveh et
al. [31], a 'replacement' of this sort would probably not correct the zinc status.
The third problem relates to the possible beneficial effect of zinc supplementa-
tion on the activity of RA. Although gross deficiency of this metal does not
occur in RA as established by Milanino et al. [13], this question cannot be
considered completely closed at the present time because of the abundant old
and recent reports suggesting that zinc is involved in the regulation of the
immune function. These data reveal new perspectives and potential indications
for zinc supplementation but also potential risks of therapeutic indications of
zinc [40]. In particular, more investigations are needed on the zinc status of the
elderly population affected with rheumatic diseases in view of the reports that a
mild zinc deficiency appears to be a significant clinical problem in elderly people
living alone [41].
COPPER
The role of copper in the treatment of inflammatory processes such as RA is of
interest for many reasons. This subject has been reviewed in the past [42-44].
128
Serum copper, as well as synovial-fluid copper, have been found to be

abnormally elevated in patients with RA [45-48]. Copper ions have a high
affinity for certain ligands and react with biological materials as well as with
some drugs used in the treatment of inflammatory arthritides and almost all
body copper is found in complexed form. Most of the copper in the plasma is
tightly bound to ceruloplasmin, a protein that behaves as an acute-phase
reactant, increasing in a non-specific fashion in response to inflammation [49].
The role of copper in inflammation has been extensively reviewed in the past
[43,44,50-52]. Most studies indicate that the increase in serum copper levels in
patients with RA is due to increased production of ceruloplasmin in response to
inflammation and not to a nutritional disturbance [53]. Confirming previous
reports on this subject, Milanino et al. reported that plasma copper and
ceruloplasmin levels in RA patients were significantly increased above normal
values, whereas the urinary excretion of the metal was not different from that of
controls [13]. With the exception of an increased urinary excretion of copper in
DPA-treated RA patients, drug therapy did not influence the copper status in
RA. Although, in previous reports the copper concentration in the cell fraction
. of blood was significantly higher in RA patients than in control subjects [54],
when blood cell copper was expressed in relation to the absolute erythrocyte
content, the level of copper in blood cells was within the normal range. Milanino
et al. [13] concluded that it seems unlikely that, as previously suggested, RA
patients have a copper deficiency [55-57]. Plasma copper has been previously
reported to correlate with disease activity [47,58], although conflicting results
have also been reported [15,46]. Although the careful study of Milanino et al.
found a significant correlation between plasma copper and the erythrocyte
sedimentation rate (ESR) and the C-reactive protein (CRP) in patients with
RA, they did not find any correlation between plasma Cu and any of the clinical
parameters [13]. They correctly suggested that plasma copper concentrations
represent a poor index of RA severity. In spite of the correlation of plasma Cu
levels with acute-phase reactants, the data reported by Milanin 0 et al. suggest
that there is no correlation with clinical parameters of disease activity [13].
D -Penicillamine, DPA, an effective copper-chelating agent, is used in the
management of RA as a second-line drug [59,60]. The chelating properties of
DPA form the basis for the use of this drug in treating Wilson's disease and
heavy metal poisoning. DPA has been reported'to be effective in the treatment
of RA, including some patients with juvenile RA [60-62].
The mechanism of action of penicillamine in RA appears to be complex [63].
Lipsky and Ziff showed that the addition of minute amounts of copper to
cultures of lymphocytes in the presence of penicillamine greatly augments the
suppression of lymphocyte responsiveness [64]. The synergistic inhibition of
lymphocyte responsiveness by penicillamine and copper salts was shown to be
restricted to helper T lymphocytes, while suppressor T cells and B lymphocytes
were not affected [65].
The striking propensity of DPA to induce autoimmune phenomena has
generated considerable interest. It has been difficult to reconcile the inhibition
129
of helper T cells by penicillamine and copper with the development of

autoimmune diseases in some patients, since an autoimmune reaction is more
likely to be evoked by a decrease in suppressor rather than in helper cell activity.
The work of Marimoto et al. that described a distinct subpopulation of cells,
within the CD4+ subset of lymphocytes, that acts as an inducer of suppressor
CD8+ cells suggested a possible explanation for this riddle [66]. Inhibition of
this CD4+ subset by the drug would be responsible for a selective decrease in
suppressor function, leading to autoimmunity.
Formation of a DPA-Cu complex may be necessary for the antirheumatic
effect of DPA. When DPA is administered to patients with RA or cystinuria,
there is a minimal depletion of copper and zinc but this is generally compen-
sated for in the normal diet [67). However, in some cases, DPA-treated patients
can develop copper deficiency [68). It has long been suspected that depletion of
copper stores by DPA may eventually lead to a relapse in DPA-treated patients.
In order to answer this question, Pullar et al. randomized 21 patients with RA
who had relapsed on DPA to copper sulphate, 20 mg daily, or matched placebo
in addition to DPA for 16 weeks [69). These authors found that, while serum
copper was increased in the copper-treated patients, no statistically significant
or clinically important improvement occurred in either group.
Bucillamine [n-2(mercapto-2-methylpropionyl)-L-cysteine] is a thiol com-
pound that differs from D -penicillamine in that it contains two free sulphydryl
groups. Clinical trials have demonstrated that its efficacy in RA is probably
comparable to that ofDPA [70). Long-term administration ofbucillamine (Buc)
in clinical trials markedly improved arthritis and suppressed the titre of
rheumatoid factor in patients with RA. Hirohata and Lipsky examined the
effects ofBuc on the in-vitro function of human B cells in comparison with those
of DPA [71]. These authors reported that, in contrast to DPA, the suppressive
activities of Buc did not require the presence of copper. Bucillamine, as well as
some of its metabolites, exerts immunosuppressive effects that are similar to
those of DPA but they also have unique effects depending on the capacity of
these compounds to form an intramolecular disulphide between their two
sulphydryl groups. Their results indicate that Buc and some of its metabolites,
inhibit cytokine production and also suppress the production of IgM at least in
part by directly inhibiting B cells. Although copper does not appear to be
necessary for the suppressive activity of bucillamine on the in-vitro function of
B cells, Yamanaka et al. have reported that DPA and Buc are able to induce
numerous DNA strand breaks in vitro upon addition of CUS04 [72]. They found
that these DNA strand breaks depend on the production of hydrogen peroxide
and were completely blocked in the presence of catalase. This phenomenon was
found to be reversible. Significant decreases in cellular NAD levels which
functionally inactivate the lymphocytes were observed at a concentration of
DPA which would damage DNA. As extensive depletion of cellular NAD
functionally inactivates lymphocytes, the formation of DNA strand breaks
may have a role in the immunomodulatory action of these antirheumatic drugs.
Bucillamine may also suppress inflammation in RA by still another pathway.
130
Eguchi et al. investigated the ability of bucillamine to prevent T-cell adhesion to

endothelial cells isolated from human umbilical vein [73]. When endothelial cells
were pretreated with bucillamine, T-cell binding was suppressed in a dose-
dependent fashion. This effect of bucillamine was antagonized by addition of
copper sulphate in vitro. The authors suggest that hydrogen peroxide generated
by bucillamine, with or without copper sulphate, may suppress inflammation,
such as that in rheumatoid synovitis, by reducing the migration of chronic
inflammatory cells from capillaries into the tissue [73]. Preliminary experiments
reported by Sawada et al. suggest that bucillamine in conjunction with copper
induces apoptosis in THP-l, a human monocyte cell line [74]. They further
speculated that this action of Buc and copper may lead to suppression of
outgrowth of cells in RA synovitis [74]. Thiopronin is a second-line drug used
to treat RA with proven efficacy in controlled trials. Sany et al. reported that
declines in serum copper and ceruloplasmin levels were not correlated with
treatment efficacy or tolerance ofthiopronine [75].
The role of copper in the host defence mechanism has not been established. It
is well-known that inflammation increases plasma levels of ceruloplasmin, an
acute-phase cuproprotein with associated antioxidant activity. Although copper
is an essential nutrient in the human diet, overt or severe deficiency is not a
major public health concern in developed countries [76-79]. However, there are
recognized factors which increase copper requirements in certain clinical
conditions. For example, it has been reported that copper deficiency occurs
secondary to gastric resection, unsupplemented total parenteral nutrition, high
levels of zinc intake or general malnutrition. The effect of chronic inflammation
on human Cu dietary requirements is unknown.
The possibility has been discussed that a pro-oxidant-antioxidant imbalance
may have a role in the pathogenesis of the rheumatic diseases. Miesel and Zuber
studied the copper-dependent antioxidant defences in inflammatory and auto-
immune rheumatic diseases [80]. These authors found that concentrations of
Cu-thionein were significantly diminished in patients with connective tissue
diseases. Corticosteroid treatment of patients with RA, systemic lupus erythe-
matosus and polymyalgia rheumatica increased the serum concentration of Cu-
thionein. These findings are of interest because this low-molecular-weight
copper protein exerts pronounced superoxide dismutase (SOD) activity and
scavenges effectively hydroxyl radicals and singlet oxygen. Copper is a necessary
co-factor of many enzymes, among them superoxide dismutase. SOD was
initially reported by Huber et al. to exhibit potent anti-inflammatory activity in
animal models of acute and chronic inflammation [81]. SODs are intracellular
enzymes that catalyse dismutation of superoxide to hydrogen peroxide and
oxygen. SODs also prevent the formation of toxic hydroly radicals and singlet
oxygen. Previous work has shown SOD to be effective in the treatment of
arthritis in horses and dogs [82-85]. However, the data suggesting the effective-
ness of intra-articular injections of SOD in the treatment of osteoarthritis and
rheumatoid arthritis are still inconclusive [86,87]. This question clearly requires
further investigation. Work in animal models of inflammation has shown that
131
inflammation can increase the amount of Cu required to maintain Cu-Zn

superoxide dismutase activity levels [88]. DiSilvestro et al. reported that Cu
supplementation (2 mg/day, 4 weeks) increased erythrocyte Cu-Zn superoxide
dismutase activity levels in 8 of 23 rheumatoid arthritis patients [89]. SOD
values were significantly lower in RA patients than in control subjects before,
but not after, supplementation. DiSilvestro et al. reported that supplementation
did not significantly affect ceruloplasmin activity. These authors suggest that
their results can be explained by a marginal Cu status in the RA patients that
they studied [89]. In relation to this question, three studies have reported
deficient intake of copper in patients with rheumatoid arthritis [18-20]. Interest
in the potential therapeutic use of copper-zinc superoxide dismutase has been
rekindled with the experimental demonstration of the beneficial effect of its use
on the articular cartilage in experimental models of arthritis [81]. Hoedt-
Schmidt et al. reported that rH-SOD exerts an inhibitory effect on the
deleterious processes on articular cartilage tissue during the course of osteoar-
thritis. They suggested that rH-SOD may counteract cartilage degradation
induced or accelerated by oxygen radicals [90]. Kakimoto et al. studied the
effect of SOD on murine collagen-induced (CIA) arthritis [91]. Among the SOD
derivatives studied, only a gelatin-SOD conjugate which has a prolonged half-
life in vivo was effective to suppress the development of CIA, while native SOD
or gelatin carrier alone were ineffective. This study found neither a significant
effect on the immune response to type II collagen nor a cellular immune
response as a consequence of treatment with gelatin-SOD. The authors suggest
that oxygen radicals may have an important role in the effector phase of the
immune response of CIA [91]. These works [90,91] are in agreement with the
previous suggestion of the involvement of superoxide radicals in inflammation
and with the role of SOD in this process [92-97].
Copper complexes of many anti-inflammatory compounds have exhibited
enhanced therapeutic effect compared with the parent ligands in several
experimental models of inflammation. Naughton et al. investigated the chemical
nature of a number of Cu(II) species present in isolated synovial fluid (SF)
samples from patients with RA [98]. This investigation was aimed at elucidating
the molecular nature of non-ceruloplasmin-bound Cu(lI) species present in
inflammatory SF samples, particularly low-molecular-mass complexes which
reportedly promote the generation of OH radical in the presence of endogenous
reductants [99]. Naughton et al. were also able to detect and quantify trace levels
(ca. 10-7 mol dm- 3) of copper ions in ultrafiltrates of SF by high-performance
liquid chromatography [98]. Circular dichroism as well as visible absorption
spectroscopic studies conducted on RA SF ultrafiltrates provided evidence for
the complexation of added eu(lI) by histidine. Cu(II)-histidinate complexes
have been shown to exhibit SOD-mimetic activity [100]. It is of interest that the
rate constant for the dismutation of O2 by Cu(II)-His species is of a similar
order of magnitude to that of SOD itself. Naughton et al. demonstrated that
further addition of Cu(II) solutions to rheumatoid SF resulted in selective
broadening of the threonine, tyrosine and phenylalanine signals, the latter two
132
aromatic amino acids being effective hydroxyl radical scavengers. Circular

dichroism and Hahn spin-echo IH NMR experiments indicate that the protein
fraction of intact SF complexes a large proportion of added Cu(II) ions. These
data are of significance in the design and screening of new Cu(II)-containing,
and potential Cu(II)-chelating agents as anti-inflammatory/antiarthritic agents
in vivo [98].
At the time of writing, there are no copper or zinc drugs approved for the
treatment of rheumatoid arthritis in the USA. Future studies on the value of
zinc and copper in the treatment of rheumatoid arthritis should probably focus
on specific pharmacological action of Cu or Zn compounds on the inflamma-
tory process or on the immune system.
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137
9
Topically applied copper preparations for
anti-inflammatory therapy
SJ Beveridge
Department of Chemistry, The University of Newcastle, Brush
Road, Ourimbah, NSW 2258, Australia
INTRODUCTION
While the recorded use of copper and its compounds for anti-inflammatory/
antiarthritic purposes goes back to the Egyptian papyri (2600 and 2200 BC), as
well as Greek and Roman writings [1], it is only in relatively recent times that
any serious attempt has been made to evaluate the potential therapeutic role of
metallic copper in inflammatory diseases. The role of copper in inflammation
continues to attract attention. This is reflected in the continued success of edited
works such as this, drawing the various interdisciplinary approaches together in
a coherent manner.
It is interesting to note that while reports on the anti-inflammatory activity of
copper compounds continue to appear in the literature (see for example [2]), the
precise role(s) of copper remains largely unexplained. Similarly, the question of
whether copper is pro- or anti-inflammatory remains, although the greater
number of reports focus on an anti-inflammatory role. Whitehouse first raised
this issue of the 'ambivalent role' of copper in inflammatory disorders in 1976
[3]. More recently, Berthon [4] revisited the question in attempting to reconcile
the capacity of copper to generate tissue-damaging hydroxyl radicals with the
observed anti-inflammatory activity. While numerous researchers have reported
a superoxide dismutase activity for copper complexes [see, for example,
Reference 5], Berthon hypothesizes that some of these complexes may exert a
physiological effect through a catalase mimetic action.
Numerous authors have reported the anti-inflammatory activity of copper
complexes, particularly those derived from non-steroidal anti-inflammatory
drugs (NSAIDs) (see for example the recent review by Sorenson [6]) when given
systemically. Reports of topically administered copper complexes remain
limited or suggest no activity [7]. However, Auer et al. [8] recently reported on
the efficacy of topically applied copper salicylate and copper phenylbutazone in
rat and horse models of acute inflammation.
It is just over 20 years ago that Walker and Keats [9] investigated the possible
139
© 1998 K/uwer Academic Publishers.
therapeutic role of metallic copper, as in the copper bracelet, by way of a double

blind trial using anodized aluminium bracelets as the placebo. In providing
some evidence for the probable therapeutic benefit from the copper bracelet,
these researchers suggested that components of sweat may act as cupriphores.
Some support for this proposal is derived from further work by Walker and
Griffin [10] who reported that 64Cu-labelled bis(glycinato) copper(II) permeated
intact cat skin. Subsequently, Odintsova reported the perfusion of intact human
skin by copper(II) acetate [11].
Additional support for a copper dissolution-skin absorption hypothesis may
be gleaned from the observation that solid, crystalline bis(glycinato) copper(II)
monohydrate can be isolated following aeration of an aqueous solution of
glycine containing metallic copper [12].
On the basis of the data obtained (as outlined above), the viability of a
transdermal route of administration for a copper-based anti-inflammatory
compound was investigated. This choice was also guided by the problems
associated with other routes of administration, such as irritancy (subcutaneous
injection), inactivity (oral) and toxicity (parenteral) [13].
To facilitate dermal absorption, it was decided to use a non-aqueous media.
This also had the advantage of stabilizing the copper salicylate complex
Cu(Hsalh-EtOH (where H 2 sal =salicylic acid), thus avoiding the hydrolysis
normally associated with Cu(Hsalh-xH20 complexes in aqueous systems. This
'carrier' system also emphasized the lipophilic character of the complex, in itself
an advantage for dermal absorption. The complex was prepared as described
previously [14], by refluxing copper hydroxide (1.0 g) with salicylic acid (7 g) in
ethanol (80 cm3) until dissolution, followed by addition of glycerol (20 cm3). Gel
formulations were also based on this preparation. The excess salicylic acid in the
preparation (5: 1 mol ratio rather than 2: 1 for complex formation) was necessary
to stabilize the complex and provide an acceptable shelf life. The copper
salicylate ethanolate complex formulation was registered as Alcusal.
This update of the 1989 publication [15] reviews the published material to
date and reports on the clinical trials undertaken to date.
TRANSCUTANEOUS ROUTE OF ADMINISTRATION

Delivery of drugs by the transcutaneous/percutaneous/transdermal route has
become well established over the past 15 years. A recent review by Bertii and
Lipsky [16] focuses primarily upon the practical aspects of transcutaneous drug
delivery. In commenting on the transcutaneous route, these workers cite
advantages including avoidance of the hepatic first-pass biotransformation and
metabolism, control of absorption and availability of multiple skin sites.
Disadvantages included the potential for skin irritancy, systemic toxicity and
the difficulty associated with the time for absorption across the dermis. The
review by Steinstrasser and Merkle [17] surveys the major factors affecting
metabolism of xenobiotics in the skin, recognizing the role of localized enzyme
activity within the skin.
140
TOPICAL COPPER FOR ANTI-INFLAMMATORY THERAPY
The suggestion that inorganic complexes may be delivered transdermally was

raised only relatively recently by Fairlie and Whitehouse [18], who reported a
range of inorganic complexes which exhibited biological activity when adminis-
tered topically. These included metal-drugs exhibiting anticancer properties or
anti-inflammatory activity. A role in the provision of nutritional supplements of
essential trace metals was also suggested.
The pharmacokinetics of topically applied salicylic acid was reported by
Singh and Roberts [19]. These workers subsequently broadened their study of
skin permeability following topical application to include a range of NSAIDs
[20].
Using a 64Cu-labelled copper salicylate ethanolate solution (prepared as
described above), it was found that the 64CU was extensively absorbed and
subsequently excreted following topical application [21].
RESULTS OF ANTI-INFLAMMATORY/ANTI-OEDEMIC ASSAYS

Although the results of animal studies used to evaluate the anti-inflammatory
(AI) lanti-oedemic activity (AO) of a range of topical copper complexes have
been summarized previously [14 and references therein], a brief review of the
results is valuable to provide the reader with a background for the human
studies which follow.
Acute AllAO activity was determined using a range of agents, including
carrageenan, zymosan and microcrystalline calcium hydroxyapatite. The animal
model was a shaved rat (various strains were employed in the numerous studies),
typically with approximately 20 cm2 of exposed dorsal skin. Because this
procedure involved exposing skin normally protected by hair, skin from the
treated area was monitored for general condition and hair regrowth over a
period of at least 4 days following the experiment. The experimental protocol
involved injection of the oedamagen into the ventral surface of both rear paws.
The diameter of the paws were measured prior to and following treatment.
The topically applied ethanolic copper(II) salicylate proved effective in
suppressing the acute inflammatory response to all of the oedemogens em-
ployed. Maximum suppression of paw oedema was consistently observed when
the oedema was initiated 6 h after applying the copper salicylate preparation.
Interestingly, there was no 'memory' effect when the oedema was initiated 24 h
after application of the copper salicylate, unlike more irritant copper complexes,
such as copper(II) bisglycinato administered subcutaneously. The acute AllAO
activity of some 30 copper(II) complexes were evaluated as part of the study,
including a number of NSAIDs, such as diflunisal and phenylbutazone. While a
number of the NSAIDs exhibited activity when applied topically, the copper
complexes showed significantly more activity. A similar result has been recently
reported by Auer et al. [8] as part of an investigation into the possible role of
topically applied copper salicylate and phenylbutazone complexes as anti-
inflammatory agents for horses.
Antiarthritic activity was evaluated using an adjuvant-induced polyarthritis.
141
COPPER AND ZINC IN INFLAMMATORY AND DEGENERAnVE DISEASES
Rats developed overt signs of polyarthritis 12 days after inoculation with the
adjuvant (delipidated Mycobacterium tuberculosis in squalene) into the base of
the tail. In this instance, changes in rear paw thickness, as well as in tail
thickness, measured following four days of drug application, were used as a
measure of antiarthritic (AIA) activity. As the duration of treatment was
significantly greater than for the AllAO studies, any signs of toxicity, such as
poor hair regrowth, weight loss etc., were especially noted. Significantly,
application of the ethanolic copper(II) salicylate complex suppressed further
development of the arthritis in all four paws and tail. Some 28 copper(II)
complexes including those of a number of NSAIDs, were also evaluated for AI
A activity using a dimethyl sulphoxide (DMSO)-glycerol vehicle. Only the
complexes of phenylbutazone and niftumic acid were more potent than the
salicylate complex, however both exhibited more toxicity than the copper
salicylate.
CLINICAL TRIALS
Results of a clinical trial with aged osteoarthritics
In a 1990 report of a double blind cross-over clinical trial of 20 elderly (63-92
years old; average 80.6 ± 7.6) arthritics [22], Alcusal gel and a placebo gel were
tested on volunteers living in an old peoples' residence. The cohort was
predominantly female (72%), with osteoarthritis the principle medical condi-
tion. Pain and mobility (or stiffness) assessments made during the use of the
particular tube (ie. Alcusal or placebo) were compared with assessments made
during the pretrial period when no trial medication was being used (current
medication was continued for the duration of the trial). Both the participants
and the consulting physicians made assessments of the condition of the
participants.
The protocol adopted for the trial involved an initial pretreatment phase,
where daily assessments of pain and mobility were recorded. At the end of this
initial phase, participants were examined by the consulting physician, and blood
and urine samples were taken for analysis. The participants were then given, and
used, a tube of gel (randomized order) twice daily for 10 days. At the end of this
first trial period, participants were again assessed by the same physician and
blood and urine samples taken. There was then a rest period of 7-14 days,
during which no trial material was applied. At the end of this rest period and
prior to commencing the second tube, participants were once again examined
by the same physician, and blood and urine samples were taken. The
participants then used the contents of tube 2, under the same instructions as
for tube 1, for 10 days. At the end of the second period, participants were once
again examined by the same physician, and blood and urine samples were taken.
A follow-up examination was made one month after the trial, with blood and
urine samples taken once again.
142
A number of biochemical parameters were examined, including the liver

enzymes y-glutamyl transpeptidase and aspartate transaminase, together with
copper and salicylate levels. Urinary samples were tested for protein.
Results of the trial were as follows:
(a) Pain assessment by participants showed a significant decrease in pain

when they were using the Alcusal gel compared with the placebo gel
(p~O.038).
(b) Assessment of pain changes by physicians indicated a significantly greater

relief for the periods when the participants were using the Alcusal gel
(p<O.OI).
(c) Based on assessments from both the participants and the physicians, a
significantly greater increase in mobility was experienced by the partici-
pants during the period when they were using the Alcusal gel than in the
period when they were using the placebo gel (p<O.023).
(d) No significant changes in blood biochemistry or in urinary protein were

detected during the period of the trials or in the follow-up examination.
There was no effect on serum copper levels.
(e) No toxic or 'upsetting' effects were observed; participants being ques-

tioned specifically on this point. Three participants reported drying of the
skin at the site of application. While such an occurrence is annoying, it can
be monitored and avoided by better compliance with the 'Directions for
use' simply by varying the site of application. This side-effect has the
'advantage' that it can be monitored and avoided by the user - unlike the
adverse gut reactions to oral aspirin and some other well-known AI drugs.
It was concluded that for the period of the trial, Alcusal gel provided
significant pain relief and a significant improvement in mobility for the
participants.
Efficacy of Alcusal in the treatment of sports injuries

In a single-tube double-blind clinical trial of the efficacy of Alcusal gel for the
relief of traumatic pain arising from sports injuries [23], a similar protocol was
used to that described above, except that the dosage regimen was 109, twice
daily for a 7day period. The biochemical parameters evaluated were the same as
for the trial described above. In this trial, there were 19 participants,
predominantly male (65%), with ages ranging from 19 to 45 years (average age
28.6±8.9 old). The most common condition of the participants was ankle
sprain, followed by tendonitis.
143
The results indicated that:
(a) Although not statistically significant, assessment by both physicians and

trial participants indicated that those who received the Alcusal gel
experienced greater pain relief during the period of the trial than those
who received the placebo gel. The definition of 'normal' improvement in
such conditions (i.e. without medical intervention), together with large
individual variability in pain score, are two factors which make it difficult
to obtain statistically significant results.
(b) The use of Alcusal gel over the period of the trial had no effect on the
biochemical parameters (including serum copper and salicylate levels) or
urinary protein. Serum copper levels in the placebo group fell by an
average of 0.41 JlmollL, while there was an average increase of 0.52
JlmollL in the Alcusal treated group. This difference was not statistically
significant.
Both clinical trials referred to above were carried out on volunteers, with full
institutional bioethical approval.
MODE OF ACTION
Previously [14, 24], it was suggested that the most likely hypothesis to account
for the activity of topically applied copper drugs was that the attached ligand
facilitated percutaneous absorption of copper(II) across the dermis, which then
acted systemically. Support for this hypothesis arose from a number of studies.
However, the observation that a high level of 64CU activity was found in the
inflammatory tissue of rats following application of 64Cu-labelled Alcusal -
suggesting that the copper had been sequestered to the inflammatory site [24] -
was particularly persuasive.
Numerous possible mechanisms have been suggested for the anti-inflamma-
tory action of copper complexes [25,26]. These include an antioxidant/SOD
mimetic reactivity, modulation of prostaglandin synthesis and histaminic
activity, and induction of lysyl oxidases. The mechanism of action of this copper
salicylate complex is unclear; however, Okuyama et al [27] have proposed that
the efficacy of copper aspirinate and copper salcylate could be due to activation
of copper-dependent opioid receptors. While this would explain the analgesic
effect, it would not explain the observed anti-inflammatory effect. Metabolic
inactivation of polymorphonuclear leucocytes has been observed with copper
salicylate, but not salicylate alone [28]. Such inhibition attenuates production of
reactive oxygen species (ROS), themselves active in tissue destruction [29].
Copper salicylate complexes have been reported to inactivate the superoxide
anion [5]. On this basis, it is suggested that inactivation of ROS and inhibition of
polymorph function contribute to the anti-inflammatory action of topically
applied copper salicylate.
144
While a recent report by Brumas et al [30] has cast doubts about the long-held
belief that copper-NSAID complexes are the active forms of the NSAIDs
themselves, the anti-inflammatory action of a range of copper complexes
suggests that the continued study of such complexes will lead to a better
understanding of the modulatory role played by copper in such diseases. At the
same time, it would be appropriate if the merits of dermal absorption were
recognized and evaluated further.
ACKNOWLEDGEMENTS
Development of a copper complex, suitable for topical application in the
treatment of inflammatory disorders, was the result of an investigation into the
biological role of copper initiated by W. Ray Walker. His insights and
perseverance are gratefully acknowledged. The opportunity to collaborate with
Michael Whitehouse over many years now has been greatly appreciated. His
immense experience and enthusiasm in the study of anti-inflammatory drugs is
invaluable.
I should also like to gratefully acknowledge valuable discussions with Dr John
Sorenson, Department of Medicinal Chemistry, College of Pharmacy, Uni-
versity of Arkansas for Medical Sciences, Little Rock, Arkansas.
I am grateful to Dr Roger Sunde, Mineral Nutrition Sciences Cluster,
University of Missouri, Columbia, Missouri, for the opportunity to spend a
semester of study leave in his Department when part of this review was written.
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146
10
Regulation by copper of rat adjuvant-
arthritis: a model of chronic inflammation
especially suitable for studying the
mechanisms of copper anti-inflammatory
activity
R Milanino1, M Marrella1, GP Velo1, P Cristofori2 and A Terron2
11nstitute of Pharmacology, University of Verona; 2Glaxo Wellcome
(Verona), Division of Medicine Safety Evaluation, Verona, Italy
INTRODUCTION
Although nowadays ignored by clinicians, the use of copper in medicine has
been a common practice for thousands of years, and the intuitive reasoning of
its importance as an 'exogenous' anti-inflammatory agent dates back to the
classic Roman age as clearly expressed, for the first time, in the Celsus' medical
book De Medicina [I]. Modern biomedical research has confirmed Celsus'
intuition, also discovering that the so-called 'endogenous" copper (i.e. the metal
naturally contained in the body) plays an important role in modulating the
inflammatory response. In fact, the hypotheses which seem possible to outline
on the basis of our knowledge today suggest that: (a) endogenous copper may
act as part of the physiological 'anti-inflammatory' replay triggered by the
organism to keep inflammation under proper control [2,3]; (b) the inflammation
is a state in which more copper is demanded and accumulated by the organism
to face the inflammatory noxa [4,5]; and (c) although inflammation is per se able
to cause an increase in the amount of endogenous copper, this defensive
response could be somehow insufficient to effectively control the inflammatory
reaction, as suggested by the fact that copper preparations have remarkable
anti-inflammatory and antiarthritic properties [6,7].
At present, over hundred copper-containing molecules, some examples of
which are shown in Table I [8-20], have been successfully tested in acute and
chronic models of animal inflammation as well as in the treatment of human
rheumatoid arthritis. In most instances, the biological activity was evident if the
copper compounds were administered parenterally, very few copper-complexes
being effective after oral dosing. This is probably due to their breakdown at the
acidic pH of the stomach [21], and to the fact that the copper ions released
147
K.D. RainsforcJ et al. (eds.). Copper and Zinc in Inflammatory and Degenerative Diseases. 147-159.
Table 1 Examples of copper preparations having anti-inflammatory or antiarthritic properties in

laboratory animals or in man
Inflammatory
Copper compound pathology Reference
Cu-Zn superoxide dismutase A, C, RA - sc, im 8

Ceruloplasmin A-iv 9
Laccase A-iv 9
Cu complexes with amino acids A-sc 10
Cu-salicylate* A-os 11
Cu-salicylate* A, C - percutaneous 12
Cu-salicylate* RA-iv 13
Cu complexes with NSAIDs other than salicylate* A, C-sc 14
Cu complexes with D-peniciIlamine A-sc 14
Cu complexes with phenols A, C - percutaneous 15
Cu complexes with benzirnidazolyl thioethers A, C-os 16
Copper thiosulphate A, C-sc 17
Copper chloride A-sc 10
Cuprous oxide A-sc 18
Copper carbonate give with the diet C-os 19
Metallic copper A -leg implant 20
A, anti-inflammatory activity in acute animal models; C, active in chronic animal models; RA, active in
rheumatoid arthritis (humans); NSAIDs, non-steroidal anti-inflammatory drugs.
The administration routes are: sc, subcutaneous; im, intramuscular; iv, intravenous
• All these complexes with anti-inflammatory drugs are significantly more active than the parent compounds
would then undergo the efficient homeostatic control of an organism which is

programmed to prevent much of the ingested copper from being accumulated
[22,23].
Nevertheless, the homeostatic regulation of copper oral absorption and
retention can be, at least in the rat, by-passed simply by sufficiently increasing
the amount of the metal administered. For example, in a preliminary experi-
ment (unpublished observation), we observed that the total copper increases by
about 11, 18 and 30% in the liver, and by 14, 26 and 33% in kidneys, after
feeding female rats for 30 days on purified diets containing, respectively, 10, 20
and 40 times the normal amount of copper (i.e. 5 ppm).
Thus, using copper-supplemented diets it seems possible to obtain copper-
enriched rats which, at least theoretically and in comparison with normal rats,
can be regarded as animals properly prepared to resist future inflammatory
challenges.
We decided to test this hypothesis by maintaining adult female rats for 58
days on diets containing 5 (controls), 50 (10 x), 100 (20 x) and 200 (40 x) ppm
of copper, and inducing an experimental arthritis 30 days after the beginning of
148
REGULATION BY COPPER OF RAT ADJUVANT-ARTHRITIS
the copper-supplemented treatments (see Figure 1). In the experiment described

here, the development ofthe pathology was monitored 14, 21 and 28 days after
administration of the inoculum. The general condition of non-inflamed animals
fed with the same copper-supplemented diets was evaluated after 30 and 58 days
of treatment, and that of inflamed animals was determined on the 28th day after
the challenge (see Figure 1).
The final results obtained as well as the possible future applications of this
model of rat adjuvant-arthritis regulated by copper, some preliminary reports of
which have already been presented at three meetings [7,19,24], are fully detailed
and discussed in this paper.
ANIMALS, MATERIALS AND METHODS

Four hundred and forty female Sprague-Dawley rats (CD-COBS from Charles
River, Italy) weighing 100 ± 6 g at the beginning of the experiments were used.
The animals were housed in groups of 5-10 with a 12-h light-dark cycle, at
constant temperature (21 ± 1°C) and humidity (55 ± 5%), and were fed ad
libitum on tap water and purified diets (Altromin DP/1000 brand, purchased
from Rieper SpA, Vandoies, Bolzano, Italy) containing 5 (control), 50, 100 and
200 ppm of copper (added as copper carbonate) and 30 ppm of zinc (added as
zinc carbonate).
ADJUVANT ARTHRITIS
INOCULUM 14d 21 d 28d
~ 1Jl
:;'
1Jl 1Jl
DIET DIET DIET DIET DIET
Od 30d 44d 51 d 58d
n U n n n
:1: ~JJL'
~ ~
1 2 2 1
2
3
Figure 1 Plan of the alimentary treatments, toxicological evaluations, and pathology monitoring
that were performed. eu contents of the diets: 5 ppm (controls), 50 ppm (10 x), 100 ppm (20 x)
and 200 ppm (40 x). 1. Full toxicological evaluation on non-inflamed rats. 2. Evaluation of the
experimental pathology (score and hind paw weights). 3. Full toxicological evaluation on inflamed
rats
149
The adjuvant arthritis was induced (after 30 days of preparatory diet) by

intradermal injection into the tail of 280 rats of 0.6 mg of heat killed
Mycobacterium butyricum finely suspended in 0.1 ml of liquid paraffin. The
arthritic score was determined on an arbitrary scale as follows: left and right
hind feet each 0-7, left and right fore feet each 0--4.5, tail 0-5, ears 0-2, eyes and
nose each 0-1 (maximum score 32).
Rats were killed in the morning between 9.00 and 10.00 [25], by exposure to
diethyl ether. Immediately after death, the blood was collected directly from the
heart using heparinized metal-free syringes. For copper and zinc analyses,
plasma and blood cells were separated by means of a clinical centrifuge; non-
haemolysed plasma was deproteinized by addition of an equal amount of 15%
trichloroacetic acid solution. Blood cells, hind paws (sectioned at the tibio-tarsal
joint) and the other solid tissues listed in Table 2 were acid-digested following
the procedure described previously [26]. Copper and zinc determinations were
performed by flame atomic absorption spectroscopy [26].
All the chemicals used were of AR grade, free from copper and zinc
impurities, and only copper- and zinc-free glassware was used [26].
Complete haematology (Table 2) was carried out following standard labora-
tory procedures. A full macroscopic examination was performed on all the
animals at death. Microscopic analysis was carried out on the tissues listed in
Table 2, after fixation and staining (haematoxylin-eosin). Forelimb tibio-tarsal
articulations were kept for 7-10 days in 10% formic acid to decalcify bones.
Samples of liver were stained with 'oil red 0' to stain for fat, and with periodic
acid Schiff (PAS) for glycogen evaluation.
Table 2 Parameters taken into account to evaluate the toxicological profile in non-inflamed and
adjuvant arthritic rats, maintained on either normal or copper-supplemented diets
Copper and zinc status Platelets Hepatic function

Plasma Count Serum aspartate aminotransferase
Whole blood cells MPV Serum alanine aminotransferase
Liver Serum alkaline phosphatase
Kidneys ESR
Spleen Determined after I & 2 h Renal function
Brain Serum creatinine
Hind paws Haematochemistry Serum urea
Sodium
White cells Potassium Histological examination
Count Chlorine Liver
Differential count Calcium Kidneys
Carbon dioxide Brain
Red cells Spinal cord
Count Blood proteins Eyes
Haematocrit Albumin Spleen, thymus, lymph nodes
Haemoglobin Total proteins Sternum, femur
MCV, MCH, MCHC Fractionated proteins Forelimb articulations
150
RESULTS AND DISCUSSION

Antiarthritic effects
As previously mentioned, the arthritis was evaluated 14, 21 and 28 days after the
challenge, scoring the animals and weighing both their hind paws.
The results of the arthritic score are summarized in Table 3. None of the Cu-
supplemented diets was able to protect the animals from the development of the
systemic reaction on day 14 and 21; on the contrary, on day 28, the 200-ppm
Cu-supplemented diet induced an inhibition of about 31% (p<0.001) in the
arthritic score. To correctly evaluate this figure, we would like to remark that, at
least in our experimental conditions, a classical 'therapeutic' treatment with
indomethacin (i.e. 2 mgkg- 1 day-l from day 14 to day 28) may reduce the
systemic reaction to the complete-adjuvant injection observed on day 28 by
about 30-40% [16].
If the hind paw weight is then considered (Figure 2), the situation is similar to
that described above, and a statistically as well as biologically significant
protection against the paw swelling (about 41 %; p < 0.001) was clearly observed
on day 28 with the 200-ppm Cu-supplemented diet.
Table 3 Effect of the alimentary regimens on the arthritic score of the rats 14, 21 and 28 days after
the challenge
Arthritic score Inhibition

Day Diet n (mean ± SD) (%)
14 Cu = 5 ppm (controls) 70 16.5±8.1

Cu=SO ppm 70 IS.8±7.9 (NS) 4.2
Cu=IOOppm 70 16.4±7.4 (NS) 0.6
Cu=200ppm 70 16.3±6.3 (NS 1.2
21 Cu = 5 ppm (controls) 50 24.8±6.4

Cu= 50 ppm 50 23.7±6.2 (NS) 4.4
Cu= 100 ppm 50 23.5 ± 6.8 (NS) 5.2
Cu=200ppm 50 24.3 ± 5.4 (NS) 2.0
28 Cu = 5 ppm (controls) 40 23.4±5.1

Cu= 50 ppm 40 21.2±6.2 (NS) 9.4
Cu= 100 ppm 40 21.8±7.1 (NS) 6.8
Cu=200 ppm 40 16.2 ±4.5*** 30.8
NS, statistically non-significant, Student's I-test; ···p<O.OOI, Student's I-test
151
3.2
•
of.
/
2.8 /
/
• *
illND PAW WEIGHT /
(g)
2.4 / ***
2 . /.
.
1.6 ----.-- '""-_._- ------ ------
14 21 28
days days days
Figure 2 Effect of alimentary treatment on the hind paw weight (average of the left and right hind
paws) of the arthritic rats 14,21 and 28 days after inoculation of complete adjuvant
• - - - . , arthritic controls (eu in the diet = 5 ppm); A - - - A, arthritic rats given a low-level
supplement (eu in the diet =50 ppm); e _. _. _e, arthritic rats given a medium-level supplement
(eu in the diet =100 ppm); + -- +, arthritic rats given a high-level supplement (eu in the
diet = 200 ppm); n=40 specimens per group (day 14),20 specimens per group (day 21),80
specimens per group (day 28); *p <0.050, ***p<O.OOI; Student's (-test
Toxicological evaluation
As mentioned above (see Animals, materials and methods), the parameters
examined to assess the toxicological profile of the rats are listed in Table 2.
They include: (a) the levels of copper and zinc, in particular to verify the
copper status in liver, kidneys and brain, which are known to be target organs
for copper toxicosis in both animals [22] and man [27], and the overall status of
152
body zinc considering that an excessive intake of copper may reduce zinc
absorption [28,29]; (b) the complete haematology and blood chemistry, particu-
larly focused on the appraisal of hepatic and renal functions; (c) the histology of
the major organs and tissues, including liver, kidneys, brain, spleen, lymph
nodes and hind paws (which are all involved in the trace element changes
induced by the copper supplement or from the pathological transformations due
to the experimental disease, or both).
Non-inflamed rats, on all four oral treatment regimens, were studied on days
30 and 58, while arthritic animals, again on all four oral treatment regimens,
were studied on day 28 after the inoculum (58 days on the diets).
Since the diet containing 200 ppm of copper was found to have a protective
effect against adjuvant arthritis, we will present and comment on the toxicolo-
gical profile relevant to this treatment group only (non-inflamed and arthritics).
However, the data observed in other treated groups (50 and 100 ppm), both
non-inflamed and arthritics, were comparable to those of the 200-ppm groups,
as far as haematology, blood chemistry and histology were concerned, and
showed a direct correlation with the dose of copper present in the diet when the
copper status was taken into account.
Toxicology of non-inflamed rats

Feeding rats with a diet containing 200 ppm of copper induced a statistically
significant accumulation of the metal, after both 30 and 58 days of treatment, in
liver, kidneys and non-inflamed hind paws, whereas no significant changes were
observed in plasma, whole blood cells and brain (Table 4). In spite of the
increased total amount of copper measured in liver and kidneys, the main
hepatic and renal function parameters of the enriched rats were within the
normal ranges (Table 4) and the histology of these organs did not reveal any sign
of toxicity at both the macroscopic and microscopic levels. Furthermore, all the
other haematological and clinical chemical indices examined (some of which are
reported in Table 4) were also within the normal range, being the minor
variations occasionally observed devoid of any pathological meaning. Once
again, the histology of the organs, other than liver and kidneys, and of the
tissues listed in Table 2 did not show any abnormal trait.
a
Finally, it is emphasized that feeding with copper-enriched diet did not
perturb the overall level of body zinc, as shown in the data reported in Table 4.
Toxicology of the arthritic rat

Besides the joint involvement, the adjuvant arthritis of the rat induces many
other pathological changes, such as liver, spleen and lymph node abnormalities,
and major variations of numerous parameters concerning the cellular, chemical
and biochemical composition of blood [30].
Also, the metabolism of both copper and zinc are markedly altered by this
experimental disease [31], being characterized, among other features, by an
153
Table 4 Some toxicological parameters measured in non-inflamed rats after 30 and 58 days on the
200-ppm copper-supplemented diet
30 days 58 days
% variation % variation
Parameter (units) (vs. controls) p (vs. controls) p
Plasma Cu (~g/ml) -4.7 NS ~5.7 NS

Blood cell (~g/ml of cells) ~7.5 NS ~2.6 NS
Liver Cu (total ~g) +28.4 <0.01 +49.5 <0.01
Kidney Cu (total ~g) +32.2 <0.01 +44.3 <0.01
Brain Cu (total ~g) ~2.6 NS +1.5 NS
Hind paw Cu (total ~g) +35.7 <0.01 + 17.3 <0.01
Plasma Zn (~/ml) ~0.8 NS +27.0 NS
Blood cell Zn (~g/ml of cells) ~4.6 NS ~5.1 NS
Liver Zn (total ~g) +5.8 NS +7.4 NS
Kidney Zn (total ~g) ~18.1 NS ~11.6 NS
Brain Zn (total ~g) +11.2 NS ~5.0 NS
Hind paw Zn (total ~g) ~1.7 NS 0.0
White blood cells (J0 3 /mm 3 ) ~16.3 NS +0.8 NS

Red blood cells (l06/ mm 3) ~1.3 NS +0.3 NS
Haemoglobin concentration (g/dl) ~2.3 <0.05 +0.2 NS
Haematocrit (%) ~0.5 NS +3.1 <0.05
Mean corpuscular volume (fL) 0.0 +2.6 NS
Total proteins (g/dl) +4.0 NS ~5.4 NS

r:J.ly ratio +8.0 NS +1.0 NS
Serum aspartate aminotransferase (U/L) ~1.7 NS +6.5 NS

Serum alanine aminotransferase (U/L) ~16.4 <0.05 ~1.7 NS
Serum alkaline phosphatase (U/L) +1.4 NS ~32.8 <0.05
Serum creatinine (mmol/L) +3.5 NS +5.7 NS

Serum urea (mmoIlL) ~5.7 NS ~3.0 NS
n = 20 rats per group; p determined by Student's I-test; NS, statistically non-significant
increase in plasma copper, a decrease in plasma zinc, and a pronounced

increase in liver copper which is usually associated with an increase in some of
the serum hepatic enzymes [30].
The overall toxicological organ, tissue and blood profiles of arthritic rats on
the normal and 200-ppm copper-supplemented diets were, however, quite
similar (data not shown).
As far as the copper and zinc status is concerned, few major variations have
been noticed (Table 5), i.e. in the copper-enriched arthritic animals, the plasma
154
Table 5 Some toxicological parameters measured in arthritic rats (28th day) either on normal
(Group 1) or 200-ppm Cu-supplemented diet (Group 2)
Group 1 Group 2 1'1%"

% variation % variation Group 1
(vs. controls of (vs. controls of vs.
Parameter (units) Group 1) Group 2) Group 2
Plasma Cu (J.lg/ml»b +110*** +98*** -11*

Plasma Zn (J.lg/ml)b -36* -21* +55*
Liver Cu (total J.lg)b + 176*** +687*** +327*"
Serum aspartate aminotransferase (U/L»C +10* +11* +9NS
Serum alanine aminotransferase (U/L»" -43*** -40*** +5NS
Serum alkaline phosphatase (u/L)C +46*" +96*" +14NS
"The differences between the two groups have been calculated on the basis of the absolute values measured, for
each parameter considered, in the arthritic rats on either normal or 200 ppm Cu-supplemented diet.
bn =35 rats per group; en =12 rats per group.
NS, statistically non-significant, ·p<0.050, ···p<O.OOI; Student's I-test
copper concentrations were found to be significantly lower and those of zinc

significantly higher than the values measured in the arthritic group maintained
on the control diet (this also confirms that the experimental pathology was,
somehow, less severe compared with that developed by the group maintained on
the standard alimentary regimen, and that the excess of copper given to the rats
seems not to have modified the normal levels of zinc absorption). Moreover, a
dramatically higher copper accumulation was observed in the liver when the
copper-enriched arthritic rats were compared with the arthritic animals kept on
standard diet, even if such an increase of hepatic copper did not appear to
influence the levels of liver enzymes measured in the serum (Table 5).
CONCLUSIONS AND FUTURE TRENDS OF RESEARCH

The data reported in this paper justify the following conclusions: (a) feeding a
40-times copper-enriched diet to the normal rat does not promote any toxic
accumulation phenomenon, at least within the 58 days covered by our
experiment; (b) on the other hand, the results obtained by challenging with
complete Freund's adjuvant the eu-enriched rat after 30 days of copper-
supplemented food clearly indicate that this experimental diet can be classified
as an 'anti-inflammatory' alimentary regimen which is both effective and devoid
of any major unwanted reactions.
At present, despite the fact that we have many data from both laboratory
animals and man suggesting a very significant potential for copper in regulating
the inflammatory process, the mode of action through which this favourable
effect is achieved has actually been more or less ignored.
155
Table 6 Some examples of possible targets of in-vivo, ex-vivo or in-vitro studies relevant to the
understanding of the toxicology of administered copper, as well as of the mechanisms of copper anti-
inflammatory and antiarthritic activities
Biomolecules Cell types

(production-expression-ac tivity) (biological behaviour)
Histamine Mast cells

Cytokines Neutrophils
Nitric oxide Monocytes
Arachidonic acid derivatives Lymphocytes
Oxygen free radicals Macrophages
Cu-ZnSOD Fibroblasts
Adhesion molecules Chondrocytes
Major histocompatibility complex proteins Endothelial cells
Nevertheless, the understanding of the mechanisms of copper anti-inflamma-

tory activity is by no means an academic exercise, being, on the contrary, essential
to a correct and efficient utilization of this trace element in therapeutics.
Inflammation is characterized by an extremely complex network of responses,
in which are implicated resident and circulating cells as well as some compo-
nents of the complement family, that, all together, respond to the inflammatory
stimulus priming a cascade of events having both local and systemic con-
sequences.
Many examples indicate that copper is actually involved in several different
biochemical and biological pathways crucial to the onset and development of
the inflammatory process [for a detailed review, see Reference 6]. For instance,
copper may reduce the release or the activity of histamine [6], may decrease the
synthesis of the 'pro-inflammatory' PGE2 concomitantly with an increase in the
production of the 'anti-inflammatory' PGF 2cx [6]. It is also important in
regulating the connective tissue metabolism [6]. Moreover, granulocyte migra-
tion [32], as well as other major functions of these cells, such as in primis oxygen
free radical production [6,33,34], are known to be modulated by copper.
In the past few years, a role has been proposed for copper also in the synthesis
and/or activity of cytokines [35], the expression and/or activity of adhesion
molecules [36] and in the pathways of nitrix oxide synthesis [37].
Finally, the well-known importance of copper in maintaining the full
efficiency of the immune response of the organism has been recently highlighted
[6,38].
Thus, from the evidence mentioned above, there do not appear to be any
certain knowledge except, perhaps, the fact that the beneficial effect of copper in
inflammation probably has a multifaceted biological base.
It is in this context that the 'rat adjuvant-arthritis subjected to regulation by
copper' model may be especially useful.
156
As a matter offact, using this model, it would be possible to carry out a wide
range of in-vivo, ex-vivo and in-vitro investigations on the production, expres-
sion and activity of many biomolecules (Table 6), and on the behaviour of
several cell types (Table 6). These research activities could, in our opinion, be
important possibly revealing some concealed, yet perhaps significant, toxicolo-
gical issue related to the problem of copper administration, as well as, of course,
elucidating the mechanisms through which copper may regulate the inflamma-
tory reaction.
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159
11
Copper and zinc compounds and cell
surface interactions
ME Davies and M Pasqualicchio
Strangeways Research Laboratory, Cambridge, UK and Instituto de
Farmacologia, Universita di Verona, Policlinico Borgo Roma, 37134
Verona, Italy
INTRODUCTION
The essential requirement of copper and zinc for normal bone and cartilage
development, and the efficacy of copper and zinc compounds as anti-inflamma-
tory and anti-arthritic agents have been well established, based primarily on a
wealth of evidence from studies of dietary copper and zinc deficiency and
supplementation in laboratory animals and man (1-5]. Surprisingly few studies
have, however, focused on the mechanisms of action of copper and zinc at the
molecular level. The aim of this brief review is to present current knowledge of
the effects of copper and zinc at the cell surface, and to discuss mechanisms
whereby modification of cellular interactions by these trace metals may playa
role in inflammatory and degenerative diseases.
EFFECTS OF COPPER AND ZINC ON IMMUNE CELL INTERACTIONS

Copper and zinc are essential trace elements which have an important function
in the normal maturation of the immune system. Animal models have shown
that deficiency of these metals results in thymic atrophy [6,7], inhibition of
antibody production [8], reduced T-cell-mediated cytotoxicity [9] and impaired
neutrophil function [10,11]. Although it is clearly established that copper and
zinc insufficiency leads to impairment of immune responsiveness [7,l2], little
information is available on the precise molecular processes involved. Recent
investigations on the effects of copper and zinc on T-cell activation [7], cytokine
production [13] and modulation of cell surface protein expression [14] have,
however, yielded some interesting data suggesting a requirement for these
metals in essential immune cell-<:ell interactions via effects on accessory surface
molecules.
161
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162
COPPER AND ZINC EFFECTS AT THE CEll.. SURFACE
Effects of copper on cell surface expression of ICAM·1

One major group of accessory molecules which is known to be essential for the
normal function of the immune system is the immunoglobulin gene superfamily.
Intercellular adhesion molecule-l (ICAM-I), a member of this superfamily is
constitutively expressed or can be induced on the surface of a wide variety of
haemopoietic and non-haemopoietic cells [15], and plays a critical role in cell-
adhesion functions in immune and inflammatory reactions [16]. These adhesive
interactions are specifically co-ordinated via a group of complementary
leucocyte ~z-integrin receptors, CDlla/CDI8 (LFA-1), CDllb/CDI8 (Mac-
1) and CDlla/CD18 (pI50.95) [17-19]. The binding of ICAM-l to the
leucointegrins regulates a wide range of short-term cell-cell interactions
including (a) adhesion and migration of leucocytes to inflammatory sites
[16,20,21], (b) antigen presentation to T cells [22], (c) T- and NK-cell-mediated
cytotoxicity [23-25], (d) metastasis of T-cell lymphomas [26], (e) phagocytosis
[27] and intracellular signalling events [28-30]. These diverse but specifically
directed cellular interactions provide a versatile array of in-vitro models to
investigate the molecular mechanisms whereby copper and zinc may participate
in inflammatory and immunological functions.
In degenerative diseases, such as rheumatoid arthritis, the inflammatory
invasion of joint tissue by immunocompetent cells is believed to involve an
active and self-perpetuating immune response [23,31,32]. Since little
information is available at the present time, it is open to speculate that copper
and zinc may exert their anti-arthritic efficacies on these cellular processes via
effects on the multifunctional cell surface adhesion molecules or their ligands.
It has recently been shown [25] that chondrocytes, the indigenous cells of
cartilage, become potential targets for invading T lymphocytes following
induction on their surface of ICAM-1 by pro-inflammatory cytokines such as
IL-l, TNFOt and IFN-y [33]. Copper sulphate and copper chloride (10 and 100
JlII1ollL) also induce expression of ICAM-1 on the surface of chondrocytes in
situ in cartilage explant cultures (Figure lA) as demonstrated by immuno-
cytochemistry [14]. Chondrocytes isolated from cartilage and grown in mono-
layer culture constitutively express a low level of ICAM-l which is markedly
upregulated by the copper salt treatment (Figure 1B and Table I). Although the
expression of ICAM-l was not quantitated, the intensity of immunostaining
induced by 100 JlmollL copper salt was estimated to be comparable to that
induced by 2 ng/ml IL-1cx [14].
Figure 1 (opposite) Expression of ICAM-I on (A) chondrocytes in normal rat cartilage and (B)
isolated pig chondrocytes cultured for 24 h in the presence, respectively, of 0.1 mmollL and 0.01
mmol/L CUS04' (C) Adhesion of pig PBMC to isolated pig chondrocytes: (II) IL-Ia (2 ng/ml)-
treated chondrocytes, untreated PBMC; (0) CUS04 (0.01 mmol/L)-treated chondrocytes, un-
treated PBMC; (f2l) CUS04 (0.01 mmollL)-treated chondrocytes, IL-Ia (2 ng/ml)-treated PBMe.
The data are means ± SE (n = 5) from 2 separate experiments. (Reproduced from Inflammophar-
macology 1995;3:35-48, Pasqualicchio, Davies and Velo, with permission of Kluwer Academic
Publishers)
163
Table 1 Upregulation of ICAM-l cell surface expression by copper and zinc and other divalent
metals
Divalent metal salt Concentration Cell type ICAM-l References
CUS04 100 ~mol/L Chondrocytes + 14

CuCh 100 ~mollL Chondrocytes + 14
CuCI 2 200 ~mollL Endothelial cells 34
CoCI 2 2mmollL Endothelial cells + 34
NiCIz 2 mmollL Endothelial cells + 34
ZnS04 10 ~mollL PBMC, Tcells 14
ZnCI 2 10 ~mollL PBMC, T cells 14
ZnS04 10 ~mollL Chondrocytes 14
ZnCI 2 2 mmol/L Endothelial cells 34
This effect of the copper ion is not species restricted. Using specific cross-
reacting antisera, copper-induced ICAM-l expression was observed in human,
rat and pig cartilage. Under the same experimental conditions, non-toxic
concentrations of zinc sulphate ( < 10 j.lmollL) did not induce ICAM-l. Copper
is not the only divalent metal to directly induce the expression of ICAM-l on
cell surfaces. Cobalt and nickel, two common contact sensitizers, have also been
reported by Goebeler et al. [34] to upregulate ICAM-l on cultured human
umbilical endothelial cells. In the same study, these authors, however, observed
no induction of ICAM-l by copper chloride (Table 1). They were also unable to
demonstrate significant upregulation of ICAM-l by nickel and cobalt on
endothelial cells in human foreskin explants, which would suggest that these
apparently conflicting differences in ICAM-l regulation are probably the result
of heterogeneity of cell types. It is also possible that cobalt, nickel and copper
induce ICAM-l by different mechanisms in different cell populations. An
autocrine mechanism involving IL-l is not believed to be responsible. Nickel
chloride appears to upregulate ICAM-I expression on cultured umbilical
endothelial cells via phorphorylation events [34]. This could well be an
important observation since it has recently been reported that protein kinase C
is involved in inflammatory mediator and cytokine-induced upregulation of
ICAM-l expression in human vascular endothelial cells [35]. Manganese has
recently been shown to induce adhesion ofT-cell hybridoma cells to ICAM-l-
expressing rat hepatocytes [26]. Upregulation ofICAM-l was not assessed but
the Mn2 +-induced activation of LFA-l was reported not to be associated with
the observed redistribution and microclustering of LFA-l. No information is
available at the present time on the mechanism whereby copper induces surface
expression ofICAM-l on chondrocytes.
It is noteworthy that the ICAM-l induced by the copper ion failed to promote
adhesion of chondrocytes to peripheral blood mononuclear cells (PBMC) even
164
COPPER AND ZINC EFFECTS AT THE CELL SURFACE
when the PBMC were activated by IL-1 [14]. Furthermore, the presence of
copper ions seemed to induce dyscohesion between these two cell types (Figure
1C). This result needs further investigation since it might provide useful
mechanistic clues. Indeed, by analogy with recent information on the mechan-
isms of other j>-integrin receptor/ligand interactions [36,37], it may be suggested
that copper ions alter receptor affinity in some way or induce unfavourable
conformational changes such that cell adhesion is disrupted or that ICAM-1 /
LFA-1ligation assumes an alternative function. It also cannot be excluded that
the induction by copper ions of an apparently non-functional ICAM-1 molecule
resulting in dyscohesion may actually be caused by perturbation of ICAM-1 /
cytoskeletal protein interactions [38], leading to disruption of intracellular
events involving signalling mechanisms.
Effects of zinc on surface expression of CD18

Immunocytochemical experiments using the monoclonal antibody TS 1/18 have
demonstrated upregulation of CO 18 on porcine (Figure 2A) and human PBMC
by zinc sulphate and zinc chloride at a non-toxic concentration of 10 IlmollL
[14, unpublished observations]. C018, the common ~2 chain of the leucocyte
integrins is present on monocytes, macrophages, neutrophils, NK cells and
cytotoxic T lymphocytes [39] and, in association with COlla (LFA-1) and
CO 11 b (Mac-I) functions as the counter-receptor for ICAM-I. LFA-1 is also
the receptor for ICAM-2 and ICAM-3 and Mac-1 binds an even broader
spectrum of unrelated ligands, including C3bi, fibrinogen, thrombospondin,
laminin, fibronectin, Leishmania promastigote gp 63 and Factor X [39,40 and
references therein]. The upregulation of COl8 expression by zinc therefore has
the potential to influence a wide range of biological activities. Our own recently
reported study [14] demonstrates that zinc significantly increases the adhesion
of porcine PBMC to autologous chondrocytes via a C018-dependent pathway
(Figure 2B). Since zinc has no effect on ICAM-1 surface expression, it seems
that the zinc-induced upregulation of C018 on PBMCs and its ligation to
ICAM-1 on chondrocytes is solely responsible for the augmented adhesive
interaction.
An alternative, but untested, pathway by which zinc might promote adhesion
between PBMCs and chondrocytes is via binding of upregulated CO 18 III b
(Mac-I) to fibronectin, which is reported to be expressed on the surface of
chondrocytes [41]. Fibronectin is a multifunctional glycoprotein which is widely
distributed in mammalian plasma, connective tissue, cell surfaces and some
basement membranes. It is capable of interacting with a variety of molecules
and cell-surface receptors [42], including Mac-Ion TNF-cx-stimulated PMNs
[40). Thus, it is obvious that any stimulatory effect of zinc on Mac-1 lfibronectin
binding will have far-reaching consequences in many cell--cell and cell-matrix
interactions.
The mechanism whereby zinc promotes cell-cell adhesion via CD 18-mediated
pathways is unknown. It is well established, however, that the Printegrins
165
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Figure 2 (A) Expression of CDI8 on pig PBMC cultured for 16 h in the presence of 0.01 mmollL
ZnS04' (B) Adhesion of untreated (11), IL-IIX (2 ng/ml)-treated ~ and ZnS04 (0.01 mmollL)-
treated ~ pig PBM C to untreated isolated pig chondrocytes and inhibition by anti-CD 18 (TS I I
18). (Reproduced from Inflammopharmacology 1995;3:35-48, Pasqualicchio, Davies and Velo,
with permission of Kluwer Academic Publishers)
166
acquire increased adhesive potential via complex intracellular signalling path-

ways triggered by a variety of stimuli, resulting in selective upregulation of
surface expression, activation and conformation modulation of these receptors.
Divalent ion-binding sites are also known to playa vital role in the regulation of
ligand binding. The binding motif, called the metal-ion-dependent adhesion site
(MIDAS), is located within the ligand-binding site of all integrin-~ chains and is
thought to form an intermediate ternary complex with the cation and the ligand,
such that cation displacement leads to a stable binding interaction [43]. The
essential requirement of Ca2+IMg2+ ions for integrin activation has been
recognized for some time. Manganese, another divalent cation, was shown to
promote cellular adhesion by increasing the affinity of the ~1-integrin CX5~1
(VLA5), probably by competing with Ca2+ or Mi+ at the cation-binding site
[44]. Indeed, Mn2+ is known to displace Ca2+ and Mg2+ from many other
receptors, including VLA3, VLA6, the vitronectin receptor, gpIIb/IIIa and
LFA-l. A variety of divalent cations has been examined for effects on the
binding offibronectin receptor to fibronectin [44]. Although Ca2+, Mg2+, Ni2+,
Cd2+ and Co2+ all enhanced attachment, Mn2+ was the most potent cation.
Cu2+ as well as Ba2+, Be2+, Fe2+, Fe3 +, Gd 3 +, La 3 +, Pb2+ and S~+ failed to
support attachment. Zinc was not included in the study. Recently, a novel role
for Mn2+ ions in adhesive function has been revealed via induction of
activation-dependent neoantigenic epitopes on Mac-1 and LFA-1, suggesting
an additional means of regulating receptor affinity [36,45].
By analogy, it would be reasonable to assume that zinc potentiates adhesion
by similar receptor-activating mechanisms. Unfortunately, few studies include
cations other than Ca2+, Mi+ and Mn2+ and thus there are, as yet, no data
available to confirm this assumption.
Effects of copper and zinc on HRG binding to T cells

A novel but rather more obscure means by which copper and zinc might
modulate immune and inflammatory processes via cell surface-glycoprotein
interactions has been reported very recently [46]. Histidine-rich glycoprotein
(HRG) is a divalent cation-binding protein with a range of activities, including
modulation of complement function and macrophage phagocytosis, and
inhibition of IL-2-dependent proliferation of T lymphocytes. Although there is
evidence that HRG binds to a 56-kDa receptor on the surface ofT cells, nothing
is known ofthe identity of the receptor.
Recent experiments using fluorescence flow cytometry revealed that zinc
sulphate (20 IlmollL) induces a 3-6-fold increase in the binding of HRG to the
human cell lines, lurkat and MT4, and the murine 010 line [46]. Copper
sulphate (10 Ilmol/L) was less effective than zinc. Binding of HRG to T cells
inhibits their adhesive capabilities. Zinc greatly potentiates the inhibitory effect
of HRG on the adhesion of murine 010 T cells to the tissue culture dish and to
ECM components, laminin, collagen and fibronectin. Interestingly, homotypic
adhesion of T cells was significantly increased by the presence of zinc,
167
suggesting involvement of an alternative adhesion pathway. Copper was without

effect in these adhesion assays.
The mechanism(s) by which zinc, and to a lesser extent copper, influence these
cell-surface interactions, albeit intriguing, is not understood at present. The
potential of zinc to modulate T-cell function in this way has obvious
implications for the regulation of inflammatory and immune reactions but
further information must be awaited before the full significance of these findings
can be assessed.
EFFECTS OF COPPER AND ZINC ON PLATELET AGGREGATION

There is now substantial evidence linking dietary copper and zinc deficiency
with defective platelet adhesion and aggregation, bleeding disorders and
impaired wound healing in several disease conditions. Recent in-vitro cell
adhesion and aggregometry studies suggest that impaired adhesion to endo-
thelial cells and increased aggregability of platelets from copper-deficient rats
(0.3 Ilg Cu/g diet) is due to decreased von Willebrand factor and increased
fibrinogen in platelets during copper deficiency [47]. In other in-vitro studies to
evaluate the effects of zinc, it was found that zinc ions added at concentrations
ranging from 50 to 500 IlmollL induce platelet aggregation mediated via surface
fibrinogen receptors associated with the major platelet integrin rllIb~3 (gp IIb/
IlIa) complex [48,49 and references therein]. Two mechanisms were examined to
explain this induction of platelet aggregation by zinc. At low concentrations (50
IlmollL), zinc is reported to potentiate the exposure of fibrinogen receptor on
the surface of ADP-activated platelets without affecting the binding of platelets
to fibrinogen. At 200-500 IlmollL (concentrations considered toxic in other cell
systems [14]), zinc causes platelet aggregation by an ADP-independent mechan-
ism [48,49]. Since (a) zinc is known to playa key role in the subcellular
distribution and activation of protein kinase C [50] and (b) this enzyme, which
contains a zinc-binding site (Zn 2+ finger-like sequence), has been implicated in
the exposure of fibrinogen receptor mediated by thrombin [51], it was concluded
[48,49] that ADP and zinc may act co-operatively to activate protein kinase C,
expose surface fibrinogen receptor and promote platelet aggregation.
Integrin-rllIb~3 does not bind any of its soluble ligands on resting circulating
platelets - a vital regulatory mechanism preventing aggregation in the absence
of the appropriate haemostatic trigger. Unactivated platelets can, however, bind
to surface-bound fibrinogen via cdIb~3, facilitating recruitment to haemostatic
events already initiated. Activation of platelets, for example by thrombin, ADP,
collagen, etc., converts rlIIb~3 into an effective receptor for a range of soluble
ligands, including fibrinogen and von Willebrand factor [43,52]. This activation
event is poorly understood, but is believed to be the result of affinity modulation
associated with conformational changes [43,52]. Divalent cations, as already
stated, are required for the majority of integrin-ligand interactions and, in the
case of rlIIb~3, the concept of a shared divalent cation and ligand-binding site is
validated by the fact that they both map within a 13-amino acid region of the ~3
168
chain which also includes a MIDAS-like domain [53]. Clearly, alterations in the
conformation of the cation/ligand binding site and displacement of cations are
likely to be important for the regulation of integrin-mediated adhesion [43).
Whether and how zinc is involved in these mechanisms is open to speculation.
CONCLUDING REMARKS
The existing data point to an interesting variety of ways in which copper and
zinc directly modulate cell surface molecules with the potential of regulating cell
function. The cell-cell and cell-matrix interactions, mediated by /3-integrin
receptors that are essential for many important physiological functions, are also
involved in pathological processes, such as tumour metastasis, haemostatic
damage and the onset and perpetuation of immune and inflammatory
responses. Although many possibilities exist for intervention by copper and zinc
in this array of integrin-mediated cellular functions, it is only by achieving a
better understanding of the molecular mechanisms involved that the potential
for new therapeutic approaches to treatment will be realised.
ACKNOWLEDGEMENTS
Our own work, carried out at Strangeways Research Laboratory, was supported
in part by Unilever Research, Port Sunlight Laboratory, UK.
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172
12
Copper and postmenopausal osteoporosis
JJ Strain
Human Nutrition Research Group, University of Ulster, Coleraine,
BT52 1SA, Northern Ireland
Osteoporosis is defined as a disease characterized by low bone mass and

microarchitectural deterioration of bone tissue leading to enhanced bone
fragility and consequent risk of fracture [1]. It is the most common metabolic
bone disease of postmenopausal women in the Western world. Like other
diseases of unknown causation, the aetiology of osteoporosis is considered to
be multifactorial. Age, genetic and reproductive history, body weight and
various life-style factors, including diet, have been associated with the disease
and can predict risk [2]. The disease may also arise as a consequence of other
diseases, e.g. Cushing's syndrome, hypogonadism and hyperparathyroidism, or
treatment, e.g. corticosteroids. Peak bone mass is achieved by the fourth decade
of life and thereafter there is a slow decrease in both sexes. Rate of bone loss in
women, however, is markedly increased around the menopause, and hormone
replacement therapy is the most effective way of slowing down this loss [3].
BIOLOGY OF BONE
The human skeleton is composed of about 20% trabecular bone and 80%
cortical bone. Trabecular bone is largely confined to the vertebrae, pelvis and
other flat bones, while cortical bone predominates in the shafts of the long
bones. The biology of bone is extremely complex [4]. Bone is constantly replaced
and remodelled by (a) osteoblasts, which are responsible for the synthesis of the
components of the extracellular matrix and for priming the matrix for its
subsequent mineralization with insoluble mineral salts, especially hydroxy-
apatite, and (b) osteoclasts, which resorb calcified bone or cartilage. Trabecular
bone contains more organic matter and is more metabolically active than
cortical bone. The accelerated bone loss which occurs around menopause in
women leads predominantly to loss of trabecular bone and frequent trabecular
perforation where both organic and mineral phases of bone are lost [5]. The loss
of normal micro architecture leads to a disproportionate loss of strength for the
amount of bone lost and osteoporotic fractures tend to occur at sites, such as the
lumbar spine, which are composed of more than 50% trabecular bone.
173
DIET AND OSTEOPOROSIS

Dietary factors other than calcium have received little attention in the aetiology
of osteoporosis. Many studies have shown that dietary calcium is important in
optimizing peak bone mass [6]. Calcium may also retard bone loss in older
postmenopausal women but appears to have little effect in prevention of the
accelerated loss of trabecular bone around the menopause [7]. Moreover, there
is an apparent 'calcium paradox' in ecological studies in that the greater the
consumption of calcium per head of population, the greater the incidence of hip
fractures [8]. It seems that bone health can be achieved on a wide range of
calcium intakes and that other life style and dietary factors, for example vitamin
D, may exert major control over both calcium metabolism and bone health [9].
DIETARY COPPER
One dietary micronutrient which might be important in bone health is copper
[10]. Osteoporotic-like bone changes are some of the most universal signs of
copper deficiency in the animal kingdom and the role of copper in bone
maintenance has been reviewed [11]. Experimental studies in rats indicate that
a dietary deficiency of copper results in bone loss, inhibition of bone growth and
formation, and promotion of pathological changes similar to those seen in
osteoporosis. Some workers have used the ovariectomized rat as a model for
postmenopausal osteoporosis and have found that the associated decreased
trabecular bone was slightly more severe with copper deficiency but was not
necessarily improved by copper repletion [12]. Osteoporosis induced by
ovariectomy, however, could be minimized by copper complexes of amine
carboxyboranes [13]. Other studies indicate that disruption of osteoblasts and
retarded bone development of young rapidly growing rats exposed to caffeine
might b~ related to decreased plasma copper levels [14] and that the effects of
other trace elements, such as manganese [15] and silicon [16], on bone structure
might be mediated via positive effects on copper utilization or metabolism.
Farm animals (sheep, cattle, horses, pigs, chickens, deer as well as dogs) with
copper deficiency, both in the field and under experimenal conditions, have bone
and cartilage abnormalities which result in thin brittle bones with increased
fragility and solubility [17-21].
Biological mechanisms
The mechanism most probably responsible for the loss of bone micro-
architecture in copper deficiency is decreased activity of the copper-dependent
enzyme, lysyl oxidase, which initiates the cross-linking in collagen and elastin
[22]. The enzyme catalyses cross-linking of newly formed collagen and tropo-
elastin fibres through the oxidative deaminations of lysine side-chains of these
proteins. This oxidation of lysine or hydroxylysine !:-amine groups, in turn, leads
to a multiplicity of spontaneous reactions of the oxidized products with other,
174
COPPER AND POSTMENOPAUSAL OSTEOPOROSIS
non-oxidized, lysine amino groups. The end result of these reactions is the
linking together of collagen or elastin strands. Lysyl oxidase is quantitatively a
major enzyme in connective tissues and may also be involved in the normal
export of copper from connective tissue cells [23].
Decreased activity of other copper-dependent enzymes, however, may also be
involved. These include superoxide dismutase, which dismutates the superoxide
radical and cytochrome oxidase which is responsible for the final transfer of
electrons to oxygen in the electron-transport chain of cell mitochondria.
Osteoclasts are known to produce superoxide and decreased activities of the
latter two enzymes (with a concomitant increase in superoxide, and possibly
peroxynitrite, production) could conceivably lead to increased bone resorption
[24). Ceruloplasmin, a multifunctional copper protein with ferroxidase activity
and copper transport functions, may also be mechanistically involved in bone
changes as this protein is increased by oestrogens which decline at the
menopause [25). Cartilage matrix glycoprotein, which has considerably
homology to ceruloplasmin and also some ceruloplasmin-like oxidase activity,
is an intracellular constituent of chondrocytes [26]. Although the function of this
protein is unknown, chondrocytes are involved in the production of cartilage
and might also playa role in the maintenance of bone health.
Human studies
Preterm infants on enteral copper-deficient feeds exhibit skeletal changes,
fractures and decreased growth [reviewed in References 25 and 27]. Moreover,
changes in the long bones are noticeable in Menkes' Disease, an inherited defect
akin to a copper-deficiency syndrome. Studies with malnourished children
indicate that copper deficiency directly affects bone organic matrix formation
and may indirectly cause decalcification through suppression of alkaline
phosphatase activity [28].
The relevance of these findings in babies and children to postmenopausal
osteoporosis is currently unclear. An observational study has suggested that low
serum copper is an additional risk factor to low dietary calcium in post-
menopausal bone loss [29]. Serum copper, however, is an unreliable index of
copper status as ceruloplasmin, the major copper protein in serum, is an acute-
phase reactant and is influenced by both inflammatory conditions and
oestrogen levels [30].
Results of two experimental studies [31,32], however, have given strong
support to the hypothesis that a low copper status might predispose to
postmenopausal osteoporosis. In one of these, the effects of supplementation
with (a) calcium (1 g Ca/d as citrate malate) and (b) a cocktail of the trace of
elements, zinc (15 mg Zn/d), manganese (5 mg Mn/d) copper (2.5 mg Cu/d) on
lumbar (L2-lA) bone mineral density (BMD) was evaluated in healthy older
(mean age 66 years) postmenopausal women, over a two-year period in a
double-blind placebo-controlled trial in San Diego, California [31].
Spinal bone loss over the two-year period in those women who took placebo
175
=
(n 18» was substantial (change in BMD from baseline -3.53 ± 1.24%). Bone
loss was slowed by supplementation with either calcium (change in BMD from
baseline -1.25 ± 1.46%, n = 13) or trace minerals (change in BMD from baseline
-1.89 ± 1.40%, n = 14) and was halted by supplementation with calcium plus
=
trace minerals (change in BMD from baseline 1.48 ± 1.40%, n 14). The latter
group was the only group which differed significantly from the placebo group
with respect to bone loss. Owing to the small sample size, there was no statistical
difference among any of the active supplement groups and it was also unclear to
what extent the observed positive effect in the group receiving calcium plus trace
minerals was dependent on all three trace elements (zinc, manganese and
copper).
Results are also available from another supplementation study involving
copper [32]. This time, younger women, aged 46-56 years, were recruited from
a general medical practice in Belfast, Northern Ireland, and were randomly
assigned double-blinded to receive either copper supplement (3 mg Cu/d as
amino acid chelate) or placebo.
Measurement of vertebral trabecular bone mineral density (VTBMD) was by
computed tomography (CT) scan at baseline and after two years. A total of 73
women entered the study, 24 took the copper supplement, 32 the placebo and 17
did not comply over the total period of the study. Those women who took the
copper supplement did not lose bone (initial VTBMD 124.6 ± 32.1 mg/cm 3 and
final VTBMD 123.8 ± 36.3 mg/cm 3 ) while those who took the placebo had
=
significantly (paired t-test, p 0.01) lower VTBMD at the end of the study
period (initial VTBMD 120.7±29.2 mg/cm 3 and final VTBMD 113.2±26.6
mg/cm 3 • Women on the copper supplement with an initial high VTBMD (most
probably the pre- and perimenopausal women) lost less bone than those who
took the placebo. There was little difference in bone lost between the groups for
women with an initial low VTBMD. There was also no difference in age, body
mass index, menopausal status, smoking, alcohol use, non-supplemental dietary
copper (about I mg/d) intakes (estimated from 10 x 24 h dietary recalls over the
study) or selected putative indices (erythrocyte copper and superoxide
dismutase) of copper status between groups at any time point.
CONCLUSIONS
Data from animal experimentation and human supplementation studies
indicate a role for dietary copper in postmenopausal osteoporosis. These data
are supported by veterinary and medical observations of osteoporotic-like bone
changes in copper-deficient animals and humans. Moreover, these changes can
be explained by a number of functional defects in copper-dependent bio-
chemical systems. Milk and dairy products are poor sources of dietary copper
and it might be prudent to recommend dietary changes to increase intake of
dietary copper by selecting more copper-rich foods, such as offal, shellfish, nuts,
seeds, wholegrain cereals, legumes, mushrooms and chocolate.
176
COPPER AND POSTMENOPAUSAL OSTEOPOROSIS
REFERENCES
1. Conference Report. Consensus Development Conference: prophylaxis and treatment of
osteoporosis. Am J Med. 1991;90:107-10.
2. Eastell R, Riggs BL. Endocrinology and aging: calcium homeostasis and osteoporosis.
Endocrinol Metab Clin N Am. 1987;16:829-42.
3. Department of Health. Advisory Group on Osteoporosis Report. London: Department of
Health; 1994.
4. Jee WSS. Introduction of skeletal function: structural and metabolic aspects. In: Bronner F,
Worrell RV, eds. A Basic Science Primer in Orthopaedics. Baltimore: Williams & Wilkins;
1991:3-34.
5. Pouilles JM, Tremollieres F, Ribot C. The effects of menopause on longitudinal bone loss
from the spine. CalcifTissue Int. 1993;52:340-3.
6. Bronner F. Calcium and osteoporosis. Am J Clin Nutr. 1994;60:831-6.
7. Dawson-Hughes B. Calcium supplementation and bone loss: a review of controlled clinical
trials. Am J Clin Nutr. 1991;54:274S-80S.
8. Hegsted DM. Calcium and osteoporosis. J Nutr. 1986;116:2316-19.
9. Sowers MF. Epidemiology of calcium and vitamin D in bone loss. J Nutr. 1993;123:413-7.
10. Strain JJ. A reassessment of diet and osteoporosis - possible role for copper. Med Hypoth.
1988;27:333-8.
11. Dollwet HHA, Sorenson JRJ. Roles of copper in bone maintenance and healing. Bioi Trace
Elem Res. 1988;18:39-48.
12. Yee CD, Kubena KS, Walker M, Champney TH, Sampson HW. The relationship of
nutritional copper to the development of postmenopausal osteoporosis in rats. Bioi Trace
Elem Res. 1995;48:1-11.
13. Rajendran KG, Sood A, Spielvogel BF, Hall IH, Norwood VM, Morse KW. Anti-
osteoporotic activity of metal-complexes of amine carboxyboranes. Appl Organometal
Chern. 1995;9:111-19.
14. Wink CS, Rossowska MJ, Nakamoto T. Effects of caffeine on bone-cells and bone-
development in fast-growing rats. Anatom Rec. 1996;1:30-8.
15. Strause LG, Hegenauer J, Salman P, Cone R, Resnick D. Effects of long-term dietary
manganese and copper deficiency on rat skeleton. 1 Nutr. 1986;116:135-41.
16. Birchall ID, Bellia IP, Roberts NB. On the mechanisms underlying the essentiality of silicon
interactions with aluminium and copper. Coord Chern Rev. 1996;149:213-40.
17. Suttle NF, Angus KW, Nisbet DI, Field AC. Osteoporosis in copper-depleted lambs. 1 Comp
Pathol. 1972;82:93-7.
18. Suttle NF, Angus KW. Effects of experimental copper deficiency on the skeleton of the calf. 1
Comp Pathol. 1978;88:137-48.
19. Pond WG, Krook LP, Klevay LM. Bone pathology without cardiovascular lesions in pigs fed
high zinc and low copper diet. Nutr Res. 1990;10:871-85.
20. Beattie JH, Avenell A. Trace elements and bone metabolism. Nutr Res Rev. 1992;5:167-88.
21. Thompson KG, Audige L, Arthur DG et al. Osteochondrosis associated with copper
deficiency in young farmed red deer and Wapiti x red deer hybrids. NZ Vet 1. 1994;42:137-
43.
22. Reiser K, McCormick RI, Rucker RB. Enzymatic and non-enzymatic cross-linking of
collagen and elastin. FASEB 1. 1992;6:2439-49.
23. Rucker RB, Romero-Chapman N, Wong T et al. Modulation of Iysyl oxidase by dietary
copper in rats. I Nutr. 1996;126:51-60.
24. Rico H. Minerals and osteoporosis. Osteoporosis Int. 1991;2:20-5.
25. Danks DM. Copper deficiency in humans. Ann Rev Nutr. 1988;8:235-57.
26. Fife RS, Moody S, Houser D, Proctor C. Studies of copper transport in cultured bovine
chondrocytes. Biochim Biophys Acta. 1994;1201:19-22.
27. Paterson CR, Burns 1. Copper deficiency in infancy. J Clin Biochem Nutr. 1988;4:175-90.
177
28. GuIer AH, Sapan N, Ediz B, Genc Z, Ozkan K. Effect of copper on liver and bone
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29. Howard G, Andon M, Bracker M, Saltman P, Strause L. Low serum copper, a risk factor
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serum caeruloplasmin activity levels. Nutr Res. 1990;10:355-8.
31. Strause L, Saltman P, Smith KT, Bracker M, Andon MB. Spinal bone loss in postmenopausal
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178
13
Menkes disease: a genetic defect of copper
transport
B. Sarkar
Department of Biochemistry Research, The Hospital for Sick
Children, Toronto, Ontario M5G 1X8, Canada
INTRODUCTION
Menkes disease was first described in 1962 [1] and involved five patients in the
same family who all died before the age of three years. They all displayed similar
clinical symptoms. Pedigree analysis revealed that the disease was limited to
males and was inherited in a sex-linked recessive manner. All patients gained
very little weight in the months following birth despite maintaining a normal
diet. Hair abnormalities were present in all the affected children. Hair appeared
coarse and brittle having an ivory-white colour due to depigmentation. Micro-
scopic analysis revealed that it was either twisted, of varying caliber or fractured
at regular intervals. Patients developed seizures between the ages of two and
fifteen months. Postmortem examination of patients showed widespread degen-
eration in the cerebrum and cerebellum, the overall brain size being significantly
smaller than average. Over the years since the first report, there has been an
increasing number of case reports conforming to the original description of the
disease. Subsequently, other clinical features were also reported, including
hypothermia, thrombosis, hyperbilirubinaemia, bone changes, arterial rupture
and characteristic facies. Menkes disease patients die before the age of three
years. In this chapter, we will discuss the biochemical, clinical, genetic and
therapeutic aspects of Menkes disease.
BIOCHEMICAL, CLINICAL AND GENETIC ASPECTS

After the original observation of the disease, the biochemical defect was
identified ten years later as an impairment in the absorption of copper [2,3].
Patients were found to have severe copper deficiency in the liver, brain and
serum accompanied by very low ceruloplasmin levels. There is drastic reduction
of intestinal absorption of copper which is considered to be responsible for the
main features of the disease [4] (Figure 1). Copper becomes trapped in the
intestinal mucosa in the form of copper metalIothionein which is excreted from
179
© 1998 KJuwer Academic Publishers.
Peripheral Tissyes
Copper Efflux
A
I
"'-
~
)~ ~ _C_P~
____ ____ --------~~~~
--~-' ~
\§ij
Portal
Circulation
Biliary
Excretion
I Low-MW
~!(--e Cu Complexes
Figure 1 Disruption of copper homeostasis in Menkes disease. In Menkes disease, absorption of

copper in the gastrointestinal tract is severely impaired. The increasing amounts of copper in the
intestinal lumen lead to the induction of metallothionein and the storage of copper as the copper-
metallothionein complex. Copper uptake and excretion by the liver are normal as are copper
enzyme levels. The severely decreased intestinal absorption of copper gives rise to a shortage of
exchangeable copper followed by a deficiency of developmentally important cuproenzymes.
Uptake by peripheral tissues is normal; however, excretion and intracellular copper trafficking
are disrupted by mutations in the Menkes copper-ATPase gene. As a result of impaired copper
efflux, peripheral tissues in Menkes patients tend to accumulate copper in the form of copper-
metallothionein. (Reproduced with permission from Reference 4)
the body when these cells are sloughed off. The lack of available copper leads to
severely decreased levels of developmentally important copper enzymes
(Table 1).
The major clinical features of the disease are the results of deficiency in
enzymes, such as lysyl oxidase, tyrosinase, cytochrome c oxidase, dopamine ~
hydroxylase, superoxide dismutase and amine oxidase. A deficiency of lysyl
oxidase, an important enzyme in the cross-linking of collagen and elastin,
results in the connective tissue defects observed in Menkes patients. Low levels
of cytochrome c oxidase can lead to temperature instability, while tyrosinase
deficiency causes the depigmentation of hair observed in affected individuals.
180
MENKES DISEASE
Table 1 Copper-containing enzymes in humans
Enzymes Reaction catalysed
Oxidoreductases
CU,Zn superoxide dismutase
(EC 1.15.1.1)
Lysyl oxidase Peptidyl-L -Iysyl-peptide+ H 20+02-+peptidyl-allysyl-

(EC 1.4.3.13) NH 3 +H 20 2
Amine oxidase
(EC 1.4.3.6)
Cytochrome c oxidase 4 ferrocytochrome C+02-+4 ferricytochrome c+2H 20

(EC 1.9.3.1)
Ceruloplasmin
(EC 1.16.3.1)
~ono-oxygenases
Tyrosinase L -Tyrosine+L -dopa +0 2-+ L-dopa +dopaquinone+ H 20
(EC 1.14.18.1)
Dopamine f3-hydroxylase Dopamine+ascorbate+02 -+ noradrenaline+

(EC 1.14.17.1) dehydroascorbate+ H 20
Peptidylglycine mono-oxygenase Peptidylglycine+ascorbate+02-+peptidyl(2-

(EC 1.14.17.3) hydroxyglycine}+dehydroascorbate+ H 20
The Menkes (MNK) gene has been mapped to the Xq 13 region of the X-
chromosome [5,6]. This information was used as a guide to isolate the gene
responsible for Menkes disease independently by three groups [7-9]. The gene
codes for a 1500-amino acid protein, predicted to be a P-type ATPase
responsible for the translocation of copper across membrane (Figure 2). The
N-terminus contains six metal-binding motifs which are highly homologous to
those found in bacterial metal transporters [10,11]. Menkes disease gene is
expressed in all tissues except the liver [7-9]. About 15-20% of the mutations in
patients with classical Menkes disease are deletions of varying sizes. Studies
have revealed nonsense, missense, deletion and splice-site mutations which lead
to exon skipping in many patients. Interestingly, several splice-site mutations
have been shown to give rise to the less severe forms of Menkes disease such as
X-linked cutis laxa and occipital horn syndrome. They are mostly characterized
by connective tissue abnormalities, such as hyperelastic skin in the former and a
bony abnormality of the skull (occipital exostoses) in the latter [12].
181
eOOH
Phosphorylation/ATP Binding
Copper Binding
Figure 2 Predicted structure of the Menkes disease copper-transporting P-type ATPase. The
predicted structure contains domains which are common to other cation transporting P-type
ATPases. In addition, the N-terminus contains six repeats of a 3D-amino acid putative copper-
binding motif. The grey transmembrane segment contains a conserved Cys-Pro-Cys motif which is
present in the transduction domain of other bacterial heavy metal ATPases. (Reproduced with
permission from Reference 4)
TREATMENT OF MENKES DISEASE

Parenteral therapy of Menkes disease with various copper salts, commencing
either before or after neurological impairment, has never convincingly shown
any significant effect on the devastating neurodegenerative progression of the
disease [13]. Earlier, copper-histidine was detected in normal human serum by
its isolation [14] and by amino acid reconstitution experiments utilizing dialysed
and undialysed sera [15]. Subsequent studies of equilibrium and kinetics of
exchange of copper between histidine and albumin revealed the physiological
importance of copper-histidine [16-18]. Human serum albumin has one strong
binding site for copper [19,20]. If copper is introduced into serum as an
inorganic salt and not as copper-histidine, it would initially stay in albumin-
bound form, making very little copper available in the low-molecular form.
Amino acids have been shown to facilitate the transport of copper. Increased
concentrations of amino acids result in an increased rate of uptake of copper in
tissues [21,22]. Histidine seems to facilitate transport of copper across cellular
membranes (hepatocytes, fibroblasts, hypothalamic tissue) whereas albumin
appears to inhibit the process [23-26]. Histidine has also been shown to enhance
the uptake of copper in human trophoblast cells in the presence of serum, a
phenomenon which is considered to be due to the release of copper that is bound
to albumin [27]. In the mixture of amino acids complexes, copper-histidine is
quantitatively the most significant [28]. Amongst all the copper amino acid
complexes, copper-histidine has the highest stability constant [29]. At physiolo-
gical pH, there is only one major complex species present with 1:2 copper-
histidine stoichiometry having a umque structural feature that is quite un-
182
MENKES DISEASE
common in other copper-amino acid complexes [30]. The results of our studies
of copper-histidine and copper transport led us to use copper-histidine in the
treatment of Menkes disease [28,31-33].
Copper histidine solution is prepared using full aseptic technique in a laminar
air flow hood under conditions that minimize damage from light. The light in
the laminar air flow hood must be turned off during copper-histidine prepara-
tion. Copper chloride is hygroscopic and should be weighed out quickly. Copper
chloride and histidine are dissolved in 0.9% sodium chloride for injection.
Vigorous stirring should be avoided as the product is sensitive to oxygen. The
solution is adjusted to pH 7.4. Final adjustment of the volume is made with
sodium chloride 0.9% injection. The copper-histidine solution is drawn up into
a sterile disposable plastic syringe and the desired aliquot filtered through a 0.22
J.lm filter into sterile glass vials. Sterility, copper analysis and pyrogen testing are
performed on each batch. Samples are stored and refrigerated in brown UV-
light-resistant bags [33].
We have used copper-histidine in solution form to treat Menkes patients
without addition of any excipients or freeze drying of the sample. It should be
noted that 'copper histidine is a relatively unstable inorganic complex and freeze
drying is a very stressful process in itself. Also, addition of a potentially reactive
excipient, such as mannitol, may interfere with copper-histidine equilibrium
since copper forms strong complexes with mannitol [34]. A slow release of
copper from this complex will result in its binding primarily to albumin which in
turn will allow very little copper to remain in its transportable form as copper-
histidine.
TREATMENT OUTCOME
In every case, plasma copper and ceruloplasmin levels have returned to normal
range within 2-3 weeks of the commencement of copper-histidine treatment. In
most cases, the levels have remained in the normal range on the same dose of
copper-histidine throughout the course of their disease. Case studies in other
laboratories have also shown normalization of serum copper, ceruloplasmin,
dopamine and norepinephrine levels upon copper-histidine treatment [35].
Regular measurements of bilirubin, alkaline phosphatase, AST and ALT have
consistently been within normal limits. Periodic measurements of BUN and
plasma creatinine and urinalysis have been consistently normal.
In our experience, the patients fall into two groups on the basis of the clinical
course of their disease. Patients, who do well neurologically, also have major
non-neurological problems attributable to Menkes disease. These patients are
intellectually normal and they never have seizures. In these patients, the
diagnosis of Menkes disease was made and copper-histidine begun early. Some
of the other patients who do poorly were diagnosed at 2-7 months. They fail to
thrive and show progressive neurological deterioration which does not appear to
be influenced significantly by the copper-histidine therapy. All have or develop
intractable seizures which are resistant to conventional anticonvulsant therapy.
183
RH MF
-J, -J,
Figure 3 Localization of mutations (arrows) in the IO-year-old (RH) and 20-year-old (MF)
patients within MNK and the corresponding protein domains. Vertical lines indicate the positions
of the introns and the exons are numbered. The predicated copper-binding domains are indicated
with Cu 1-6 and the transmembrane domains with vertical black bars. PD phosphatase domain;
CC, cation channel; D, phosphorylation domain; ATP, ATP-binding domain (Reproduced with
permission from Reference 36)
We have characterized the genetic defects in two patients (a 20 year old and a
10 year old) who were treated early with copper-histidine solution by injection
[36]. Using a combination of single-strand conformation analysis and direct
sequencing of amplified exons, a single base-pair deletion was detected in exon 4
in one patient and in exon 12 in the other (Figure 3). Both mutations lead to a
frameshift and create a premature termination codon within the same exon. In
the 20-year-old patient, the mutation resides in exon 12 at codon 836, producing
a termination codon upstream of the phosphatase domain. This protein product
would lack the ATPase 'core' except for the first 4 transmembrane domains. In
the 10-year-old patient, the mutation lies in exon 4 at codon 297, introducing a
termination codon within the third metal-binding domain; the resultant
polypeptide would lack the entire ATPase 'core'. The severity of these mutations
indicates that both patients have the lethal form of Menkes disease and lends
support to the efficacy of their early copper-histidine therapy, especially in
correcting the severe neurological symptoms.
DISCUSSION
In Menkes disease, copper-histidine therapy appears to be effective in prevent-
ing severe neurodegenerative problems when the treatment is initiated very early
in life. Many of the clinical symptoms of Menkes disease are attributable to the
lack of copper availability for copper-dependent enzymes (Table 1). Paradoxi-
cally, one finds increased copper levels in most hepatic tissues of Menkes
patients and in cultured fibroblasts. The suggestion, based on sequence data,
that the Menkes gene product is involved in egress rather than ingress fits well
with the observation that cultured Menkes fibroblasts take up copper with
normal kinetics but are deficient in efflux [37]. The excess copper in Menkes
disease is mostly bound to metallothionein in different array of cells [37].
184
MENKES DISEASE
The low levels of copper in liver and brain and the accumulation of copper in
placenta of fetuses affected with Menkes disease indicate a very early onset of
the biochemical derangement responsible for the disease. The efficacy of early
treatment of copper-histidine may be the result of making copper available
during the critical period of myelination and development of the central nervous
system. The normal human neonate has reduced serum copper and serum
ceruloplasmin [38].
The mechanism of the efficacy of copper-histidine treatment in Menkes
disease is not clear. Copper-histidine may bypass the defective efflux mechan-
ism and avoid trapping in the cell. Alternatively, it may overcome the cellular
defect in the incorporation of copper into copper enzymes. Albumin binds
copper avidly and any form of inorganic copper salt which readily attaches to
albumin has been shown to inhibit copper transport in cells. From the
thermodynamic point of view, copper-histidine keeps copper away from
albumin. Copper-histidine is a physiological form of copper [14] and might be
the physiological form in which copper crosses the blood-brain barrier. It has
been shown in vitro that copper histidine uptake occurs in mammalian brain
and that histidine facilitates copper uptake under certain conditions [39].
Despite significant improvements due to the early copper-histidine treatment
of Menkes disease patients, connective tissue disorders continue to persist,
indicating that lysyl oxidase levels are not restored by copper-histidine injec-
tion. It is possible that copper presented in this form is unavailable for
incorporation into lysyl oxidase. The oxidation state of copper may affect its
mobility into certain intracellular compartments. Further research in this area
should explore treatment with both copper(I) and copper(II) which may be
more effective than either alone.
ACKNOWLEDGEMENTS
Research was supported by the Medical Research Council of Canada.
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187
Index
accessory Zn-proteins 10 antioxidant(s) 13, 86

acetaldehyde 86 activity 93
acetate 27(table) apo-copper-proteins 36
like Cu(II) complexes 29(fig.) apoferritin 13
type copper complexes 31 apoprotein 67
acetylsalicylic acid 29 apoptosis 93(table), 103-4
acid-pepsin secretion 86 apothionein 72
acne 90-1 arachidonate metabolism 123
acrodermatitis enteropathica 4, 128 archaebacteria 48
actin 10 arthritis, topical zinc 89-90
adductin 10 ascorbic acid 12
adhesion molecules 156 Asp-Ala-His 26
adjuvent arthritis 82 Asp-Ala-His-NMA 27(table)
adrenalectomy 103 aspartate transcarbamylase 56
Adversariorum varii argumenti aspirin 94, 119, 121
albumin 8 asthmatic conditions 97
binding zinc 81 astringent 91
alcohol dehydrogenase 50, 55 astrocytes 64, 70
alcoholic liver disease, chronic 84 atomic absorption spectroscopy 80-1
Alcusal 140, 142-4 ATPase "core" 184
CX2 globulins 81 autosomal recessive copper toxicosis 3
cxrmacroglobulin 8
cx-helix macrodipoles 52 B-Iymphocytes 72
aluminium 86 bacteriophage T4 helix, destabilizing
Alzheimer's disease 64, 70, 86-7 protein 56
American Academy of Paediatrics, Committee 2-benzoylpyridine 3 I (fig.)
on Drugs 4 ~-alanyl-L -histidine-zinc 83
amine oxidase 22(table), 180 "bidentate" site conformation 52
amino acids 8 bile 13
aminopyrine-N-demethylase 119 bone 9
amyloid 86 biology 173
~~ 87 collagen 14
precursor protein 87 growth, copper deficiency 14
anaemia, zinc-induced 4 metabolism 83
androgens II osteoporotic like changes 174
aniline-p-hydroxylase 119 resorptive activity 83
anorexia 3,96 trabecular 173-4
antibody response 14 BSA 27(table)
anticancer drugs 71 bucillamine 130-1
antigastric-ulcer activity 93 buthionine sulphoxamine 36
antinuclear agents 27-8
189
C3bi 165 results of assays 141-2

C-reactive protein 129 transcutaneous 140-1
cadmium 61 antiarthritic effects 151-2
calcium binding site of human serum
ion flux 72 albumin 24(fig.)
paradox 174 bleomycin 115
calmodulin 97 bracelets 140
carbon dioxide 56 cell surface expression ofICAM-l,
carbon tetrachloride 67 effects 163-5
carbonic anhydrase 47,55 chelates
isoenzyme II 85 formation in vivo 120-1
carboxylate-His-Zn 52 new anti-inflammation 11920
carboxypeptidase A 52, 53 (table), 55(fig.), SOD-mimic activity 118-19
56-7 chloride 183
cardiac function 14 complexes 21-6
cardiovascular integrity 14 catalytic activity 21
carrageenan paw oedema 119 lipophilic 26
carrageenan-induced pleurisy 90 redox behaviours 25-6
cartilage matrix glycoprotein 175 stability 21-5
cathepsin B 67 therapy 113-24
CDI8 165-7 compounds 19-38
Cd-resistant clones 71 biochemistry 30-8
cell differentiation 66 selected 26-30
cell surface protein expression, copper containing molecules 147-8
effect 161, 163-5 deficiency 4, 14
cellular immune effects 97-102 syndromes 175
ceruloplasmin 9,12-13,21-4,114-15,129-32, dependent amino oxidase 20(fig.)
175 dependent transcriptional activator 27
gene 126 dietary 174-6
cervical carcinoma 114 dissolution-skin absorption
charge density 49 hypothesis 140
chelating molecules 72 effect on:
chemotaxis, neutrophil 90 HRG binding to T-cells 167-8
chief-cells 95 platelet aggregation 168-9
cholesterol 14 electron donor 79
chromatin 48 "endogenous" 147
chromosome-16 63 enzymes 13-14
cimetidine 27(table), 28, 91 excretion 12-13
circular dichroism 133 functional roles 13-14
cis-acting DNA sequences 63 histidine solution 183-4
cis-platin 71 inflammation, biodistribution 120
cobalt 164 metabolism 13
cognitive functions 87 metallothionein 179
colitis 83 neonate 185
collagen 83, 174 non-steroidal anti-inflammatory drugs 28
concanavalin A 14 oral absorption 148
copper osteoporosis, postmenopausal 173-6
absorption 12-13,69 oxides 19
acetates 19 oxygen biochemistry, roles 20(fig.)
ancient medicine 19-21 proteins, mammalian 22-3(tables)
anti-inflammatory therapy, topically responsive leucopenia 4
applied 139-45 rheumatic disease, treatment 128-33
clinical trials 142-4 salicylate ethanol ate complex 140-1
mode of action 144-5 stress protein 23(table)
190
INDEX
sulphate 19 Cu(II)(3,4,7 ,8-tetramethyl-l, 10-

sulphide 19 phenanthroline) 115
supplemented diets I 50 (table) Cu(I)-thionein 34(table)
thiolate luminescence 25 Cu(I1)(2,3,4-trihydroxybenzaldoxy-
thionein 27 imate) 115
tissue distribution 12-13 Cu(tyr)z 33-4(tables)
toxicological evaluation 152-5 Cu,Zn-SOD 23(table), 30, 31, 33(table)
toxicosis 3 cumene hydroperoxide 36
transport 12-13 cupric acetate, anti-inflammatory activity 20
copper-diSchiffbase complexes 25,30,36- cuprifores 140
7(tables) Cushing's syndrome 173
CU,Zn-SOD 35(table) cyanobacteria 72
magnetic behaviour and electronic cyclic voltametry 25
absorption 35(table) cyclophosphamide 90, 96
cortex II cyclosporine A 90
Crohn's disease 83 Cys thiolate ligands 56
Cu(I) / Cu(II) 49 Cys-Cys arrangement 62
CU2+(aq) 34(table) Cys-thiolate 50
Cu(acetylsalicylate) 33(table), 34(table) Cys-X-Cys arrangement 62
Cu(II)(acetylsalicylate) 116 cysteine 9
Cu(II) amino acids lIS rich intestinal protein 8, 69
Cu[cyclo(His)2] 33(table) cytochrome C oxidase 22(table), 115, 180
Cu-deficient diet 66 cytochrome oxidase 20(fig.)
Cu-dependent antioxidant 67 cytochrome P-450 mono-oxygenase 123
Cu(di-isopropylsalicylate) 33(table), 34(table) cytokines 71, 156
Cu(II)(dimethylglyoxime) lIS cytoprotective effects 94
Cu-EDTA 33(table) cytosol 67
Cu(formate) 34(table)
Cu(glu-his-Ieu) 34(table) d-d-absorption maxima 25
Cu(gly-his) 34(table) D-penicillamine 129
Cu(II)(glycyl-glycyl-histidinate) lIS daunorubicin 117
Cu(II)(glycyl-histidyl-Iysine) lIS dehalogenation 27
Cu(I)-GSH 68-9 delayed-type hypersensitivity 96
Cu(indomethacin) 28, 34(table) desquamation 9, 70
Cu(II)(2-keto-3-etoxybutyraldehyde-bis- dexamethasone 66-7
thiosemicarbazone) lIS diclofenac 30, 91
Cu(1ys)z 33-4(tables) diethylmaleate 36
Cu(I) MT-GSH 68-9 digestive secretion 9
Cu(I)-orthophenanthroline 21 4-dimethylaminoazobenzene 115
Cu-penicillamine 34(table) dioxygen transport 20(fig.)
Cu(II)-phenanthroline 25 DNA polymerase 10
Cu-Pulm 33(table) DNA replication 69
Cu-PuPhePy 30, 33-4(tables) DNA-binding domains 10
Cu-PuPy 30, 33(table), 34(table) DNA-dependent RNA-polymerase 10
Cu(II)(pyridine-2-carboxaldehyde-2- dopamine ~-hydroxylase 180
pyridylhydrazonate) 115 dopamine ~-mono-oxygenases 20(fig.),
Cu(II)(pyruvaldehyde-bis- 22(tab1e)
thiosemicarbazone) 115 dwarfism 3
Cu(II)(salycyl-aldehydebenzoyl-
hydrasonate) lIS Ehrlich ascites lIS
Cu(salicylate) 33(table), 34(table) tumour 96
Cu(II)-serum albumin, BSA 25 Ehrlich carcinomas 116
Cu-TAAB 33-4(tables) eicosanoid
metabolism 93(table)
191
eicosanoid (continued) gastroduodenal mucosal damage 92

synthesis, modulation 123 gastrointestinal excretion 9
elastin 174 Gaubius 1-2,4
electron gene regulatory elements 71
paramagnetic resonance 25 gene-regulatory proteins 79
spin resonance studies 116 genetic information 48
transfer reaction 19 glucagon 8-9, 13
transport 13 glucocorticoids 8-9, II, 13
chains 25 responsive hMT-J-E gene 64
electrophilic strain distortion 52 glutamatergic neurons 69
electrostatic glutathione 13,26, 93(table)
effects 52 disulphide 68
potential 49 Gly-Gly-His 26, 27(table)
endothelial cells 8, 10 glycine 27(table)
endotoxins 9 glycocalyx 8
energy dispersive X-ray microanalysis 83 growth hormone-prolactin receptor
energy metabolic changes 93(table) complex 56
energy-independent carrier-mediated growth inhibitory factor 64
process 8 growth retardation 3, 96
entropy 51
epinephrine 8-9, 13 H2-receptor antagonists 91
erythrocyte 8, 50 H-bond networks 52
ghosts 10 haemocyanin 27
erythroleukaemia cells 34 haemoglobin 126
estradiol 14 Hahn spin-echo 133
ethanol 56, 94 heavy-metal transporter ATPases 72
exon-12 184 Helicobacter pylori 93(table)
heparin 26
factor V 23(table) herpetic infections 97
famotidine 28 Herpin 3
Fe(II)/Fe(III) 49 hippocampus 11
female gonadal function 14 His-imidazolyl ring 52
Fenton reactions 20(fig.), 25 histamine 27(table), 93(table), 97
ferroxidase 13 release 86
fibrinogen 165 histidine 8-9, 27(table), 132
receptor 168 imidazole-nitro gens 30
fibronectin 165 rich glycoprotein 167-8
flores zinci 2 hMT-2 gene 64
flurbiprofen 29 Hodgkin's disease 114
formate 27(table) homeostatic mechanism 14
I-formylisoquinolone hormone(s) 11-12
thiosemicarbazone 115 receptor proteins 10
2-formylpyridine 115 host defence mechanism 128
fractures, osteoporotic 173 HSA 27(table)
free radical 10 hydrogen peroxide 28
stress 38
GABA-mediated synaptic transmission II hydrolases 79
Gal-4-domain 56 hydroxyl radical(s) 20-1, 28, 139
galactose oxidase 20(fig.) scavengers 133
gall bladder 13 hydroxylysine-E-amine groups 174
y-globulin 123 hyper bilirubinaemia 179
y-glutamyl cysteinyl 61 hypercupraemia 9
gastric secretory volume 85 hyperparathyroidism 173
gastric ulcer disease 91 hypochlorite 28
192
INDEX
hypogeusia 3 leukotriene(s} 93(table}

hypogonadism 3,11,173 Lewis acid 50
hypothermia 179 ligand(s} 7
hypoxanthine 27(table} field effect 51
hypozincaemia 9 ligases 79
lipid peroxidation 13, 123
imidazolate 30 lipophilic metal complexes 89
imidazole receptor 28 lipopolysaccharide 66, 104
immune cell interactions 5-lipoxygenase activity 94
copper, effects 161-9 liver 12-13
zinc, effects 161-9 cells 8
immune-deficient states 96 cirrhosis 128
immunocompetence 14 copper storage and excretion 13
immunoglobulin M secretion 72 zinc uptake 8
indomethacin 29(fig.},94, 119 lonacolac 29(fig.}
inflammatory bowel diseases 83-4 Ludemann I
inflammed synovia 82 Luna fixa Ludemanni
inner-sphere water exchange 51 luteinizing hormone-releasing hormone
insulin II secretion 123
allosteric behaviour 56 Iyases 79
hexamer 56 lymphocyte, phytohaemoagglutinin 14
integrin cxII~b~-3 168 lymphoid atrophy 128
integrin-ligand interactions 168 Iymphokines 66
intercellular adhesion molecule lysosomal exocytosis 13
(ICAM-l) 163-5 lysosomal membrane 123
interleukin-I 66,83, 102, 126 Iysosomes 12, 67
interleukin-2 83,96 Iysyloxidase 22(table}, liS, 123, 174,
interleukin-6 83 181(table}
intestinal absorption 69
intestinal mucosa 8 malabsorption syndrome 128
intoxications 3 malachite 19
intracellular compartment 9 mammalian tissues, adult 65
ionic radii for tetraco-ordinate manganese 174
complex 49(table} matrix metalloproteases 51
isometallothioneins 62 matrix metalloproteinases 57
membrane skeleton protein 10
lahn-Teller-distorted H 2 0 ligands 31 Menke's disease 13, 72, 175, 179-85
K-562 erythroleukaemia cells 116 biochemical, clinical and genetic
Kayser-Fleischer rings 3 aspects 179-81
keratinization 14 treatment 182-3
ketoprofen 30, 92 outcome 183-4
kidney, cortex 9 menopause 173-4
metabolism, intermediary 47
I-amino acids 12 metal
L-carnosine 94 acceptor 68
I-histidine 12 binding factor 64
laminin 165 chelation 52
leg ulcers 88 detoxification I3, 72
leguminous proteins 12 donor 68
Leishmania 165 fume fever 4
Lenthauser 115-16 homeostasis 70
leucocytes 8 ion-dependent adhesion site 167
leukaemia, acute 114 reserves 68
leukopaenia 96 response elements 63
193
metal (continued) neuroprotection 70

responsive factor 64 nickel 164
thiolate polypeptides 61 nicotinamide-dinucleotide redox ratios 38
trans cellular factor 64 niflumic acid 142
transporter 68 nitric oxide 27,28,31-2,71
metalloenzymes 48 synthesis 156
metallothioneins 8, 23(table), 61-73, 79 nitrite 27
biosynthesis 61,63-5 nitrite reductase 27
content 65-7 nitro-blue tetrazolium chloride 21
cytoprotective role 71 nitrosyl copper complexes 27
degradation 67 nitroxybutylesters 29
hepatic 82 nitroxyl anion 32
immune system 71-3 NK-cell activity II
induction 65-7 NMR 47
isoforms 62-3 non-ceruloplasmin-bound 132
location 65-7 non-enzymatic Cu proteins 13
role 68-71 non-essential metals 70
structures 62-3 non-Hodgkin's lymphoma 114
tissue specific 62 non-saturable transport 8
translocation 66 non-steroidal anti-inflammatory drugs 29-
turnover 67 30, 66, 87, 126
types 62 NUC-18 103
metzincins 53(table), 55(fig,), 57 nucleic acid(s) 21
Michaelis-Menten kinetics 94 replication 10
micronutrients 96 nucleophilic reactions 52
mitogens 72 nucleotides 26
mono-oxygenase, activation 27
mono-oxygenases, down-regulation 119 oesophageal irritation 91
monocytes 71 oesophagitis 85
monokines 104 oestrogens II
monophenol mono-oxygenase 23 (table) opioid peptides I J
mRNA MT-3 70 osmotic fragility 10
MTIII-mRNA 69 osteoarthritis 131
mucosal membrane "stabilization" 93(table) osteoblastic activity 83
mucosal protection 86 osteoporosis 83
mucus production 93(table) postmenopausal 173-6
Mycobacterium butyricum 150 bone 173
Mycobacterium tuberculosis 142 diet l74
myelomas 114 osteosarcoma 114
oxidative dealkylation 27
N-methyl-D -aspartate receptor J I oxido-reductases 79
N-terminal copper binding site 26 2-oxoglutarate-dependent hydroxylase 123
NADPH-dependent P-450 reductase 123 oxygen radicals 71, 132
naproxen 91 oxygen transport 13
Naturalis Historia 19 oxyradical production 93(table)
nervous system 13
functions J 1 Papyrus Ebers 19
neural signalling process II Paracelsus 1-2
neurocuprein 23(table) penicillamine 28
neuroendocrine abnormalities 96 pepsin 93(table)
neuroendocrine axis 95 peptide hormones 48
neurofibrilary tangles 86 peptidylglycine mono-oxygenase 22(table)
neuronal signals 69 peroxidated lipids 10
neurone growth inhibitory activity 70 peroxynitrite 32(fig,)
194
INDEX
pH-dependent dissociation 94 albumin levels in plasma 81

phagocytosis 90 therapy
phenylbutazone 119, 142 copper 128-33
phosphate-His-Zn network 52 corticosteroids 131
phospholipase C 79 ZInC 80-2, 88, 125-8
phytate(s) 7,26, 84-5 rhinovirus 97
phytomitogens 11
pilocarpine 70 sal vitrioli
pinocytosis 70 salicylate(s) 27(table), 29(fig.)
piroxicam 91 salicylidine-anthranilic acid Schiff bases 119
pKa modulation 52 scavenging properties 13
platelet aggregation 168-9 Schouwink 3
defective II Schroeder van der Kolk 2
Pliny the Elder 19 second messenger system 104
Polaprezinc 91 semen 25
polyadenylation signal-AATAA 63 sexual development, delayed 3
poly(ADP-ribose) polymerase 38 3d-shell electrons 49
polyarthritis 142 sickle cell disease 128
polyclonal activators 42 silicon 174
polymorphonuclear leucocyte(s) 28, 126 singlet oxygen 28
activities, modulation 121-2 skeletal growth 11
postmenopausal women 173 skin
postsynaptic effects 11 desquamation 9
Prasad-Halstead syndrome 84 infections 91
pre-prothrombincarboxylase 123 slow-release trace-metal complexes 89
proaccelerin 23(table) smoke bombs 4
proenzyme activation 51 smooth muscle 97
progesterone 11 SOD-mimicking substances 30
prostaglandin 11, 123 sodium/amino acid co-transport 9
D2 86 somatomedin C II
E 93(table) spectin 10
E2 102, 123 spectroscopic "silence" 47
protein folding 51 spinal cord, myelinization 14
protein kinase C 56, 164, 168 spontaneous dismutation 34(table)
acti vi ty 122 5-SRNA gene transcription 10
protein metal site 52 static magnetic field 67
protein-mediated transport 12 steroid
protein-protein interactions 51, 56 receptors 12, 79
proteoglycan synthesis 83 thyroid 56
psoriatic arthritis 81-2 stress 85
Pulm 27(table) acute-phase response 66
pulse-radiolytically generated superoxide 21 conditions 8
PuPhePy 27(table) induced gastric lesions 86
PuPy 27(table) oxidative 71
putrescine 30, 31 (fig.) sulphydryl reactivity 93(table)
pyridine-2-aldehyde 31 (fig,) superoxide 28, 32(fig.)
pyrithione 103 superoxide dismutase 10, 20(fig.), 21, 49, 67,
79,114.131-2,180
radical oxygen scavengers 66 sweat 9
ranitidine 91 synovial fluid 132
reactive oxygen species 28, 144-5 synovial tissue(s) 81-2,85
reduction kinetics 25
reserpine-induced ulcers 95 T-cell 10-11
rheumatoid arthritis 28, 85 mediated cytotoxicity 161
195
T-helper dysfunction 128 C 84,86

TAAB 27(table) D 174
telencephalon 69 E 86
tetrachlorocupratechelates 119 vitronectin 167
Theophrastus Bombastus von Hohenheim VLA-3 167
thermolysin 53(table), 55(fig.), 56-7 VLA-6 167
thiols 10 von Wille brand factor 168
thiopronin 13 I
thrombin 168 Wilson's disease 3, 13
thrombosis 179 wound healing, impaired 3
thrombospondin 165 wounds 91
thromboxane 123
B2 86 X-linked cutis laxa 181
thy-I lymphocytes 83 X-ray crystallography 47
thymocytes 103 X-ray emission nuclear microprobe 81
thymulin 79 X-ray fluorescence spectrophotometry 81
thymus 96 xanthine/xanthine oxidase system 21
atrophy 103 xanthine oxidase-acetaldehyde system 71
hormones II
tissue pigmentation 14 Z-103 91
tonsilitis 19-20 zmc
total parenteral nutrition 96, 131 acetate 96
TPEN 103 acexamate 91-3
trabecular bone mineral density 176 Alzheimer's disease 87
trace-element metabolism II amino acid complexes 95
trans-acting factors 63-4 antiarthritic activity 102
transcription factors 10, 56 anti tumour effects 96-7
transcuprein 24 antiviral effects 97
transcytotic vesicles 8 apoptosis 103-4
transferrin 13 as:
transmucosal antidote for intoxications 3
flux 9 antiepileptic 2-3
transport 8 emetic 3
triamcinolone 119 aspartate 95
trigl ycerides 14 binding proteins 8
trigonal biprism 63 bioinorganic chemistry 48-51
tubulin polymerization 10 biological space 47-8
tumour cells 38 bound water 52
tumour necrosis factor-a 83, 102 brain 86-7
tymulin II carnosine 91, 93-4
tyrosinase 20(fig.), 180 challenge test 87
tyrosine phosphorylation 102 chelating agents 87
chloride 90, 95
undecylenic acid, fungicidal properties 90 cancer induced 4
upper gastrointestinal ulceration 85-6 cimetidine complex 95
uracil 27(table) co-ordinate bonds 50
UVB irradiation 67 co-ordination and function in proteins 51-
6
vascular permeability 90 co-ordination geometry, dynamic 51
verdigris 19-20 co-ordination polyhedron 56
vesicular zinc pools 69 compounds
viral proteins 56 antiulcer activity, mechanisms 93(table)
vitamin(s) lipid peroxidation 99(table)
A 86 lysosomal enzyme release 98(table)
196
INDEX
mast-cell degranulation 98(table) physiological properties 7-12

mononuclear/cytokine reactions 100- absorption 7-9
1(tables) excretion 7-9
oxygen consumption 99(table) functional roles 10-12
reactive oxygen species 99(table) tissue distribution 7-9
deficiency 96, 128 transport 7-9
blood cell components 11 plasma levels 81, 86
dietary 10 proteases 56-7
immune function 10 chemistry 53(table)
primary or subclinical 84-5 rheumatic disease, treatment 125-8
syndromes 3 selection during evolution 48
therapy 80-104 slow release forms 87
depletion 8 sulphadiazines 91
diet 127 sulphate 1,3,83,91
deficient 103 supplementation 3-4,96, 128
effect on: surface expression ofCD-18, effects 165-7
HRG binding to T-cells 167-8 therapy
platelet aggregation 168-9 gastrointestinal diseases 88-104
elderly population 128 history 1-4
finger 52 inflammatory disease 88-104
transcription factors 63 rationale 80-7
glycinate 95 topical, arthritis
hepatic 86 anti-inflammatory 90-1
homeostasis 9, 86 antimicrobial effects 90-1
leucocyte 82 wound healing 90-1
malabsorption 127 toxicity 4
monoglycerolate 89-90, 94-5 undecylenate 90
neuromodulation 69 urinary elimination 81
oxide 1-2, 90-1 Zn-Mt-I 67
side effects 4 Zn(II) oxidation 49
197

1998 - Copper and Zinc in Inflammatory and Degenerative Diseases

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1998 - Copper and Zinc in Inflammatory and Degenerative Diseases

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Copper and Zinc in Inflammatory and

ISBN 978-94-010-5757-8 ISBN 978-94-011-3963-2 (eBook)

Printed an acid-free paper

AII Rights Reserved

List of Contributors vii

JRJ SORENSON UWESER

(For the editors)

HISTORY OF ZINC THERAPY

~.d.LT£lUVS NON SIT.QYl SVVS USE. POT.ESr.

>4hVl\£OLl THEOPHRASTI AS HOHE.NHAI M

Figure 1 Paracelsus at the age of 45. Engraving by Augustin Hirschvogel, 1538

zinc sulphate. This is approximately 13 times the dose we prescribe nowadays

ZINC AS EMETIC AND ANTIDOTE FOR INTOXICATIONS

ZINC TO TREAT COPPER TOXICOSIS IN WILSON'S DISEASE

ZINC THERAPY FOR ZINC DEFICIENCY SYNDROMES

ZINC AS A DRUG FOR ACRODERMATITIS ENTEROPATHICA

TOXICITY OF ZINC: COPPER-RESPONSIVE LEUCOPENIA AND

or histidine via a sodium/amino acid co-transport mechanism [14].

the response of T-lymphocytes to phytomitogens, in T-cell-dependent antibody

to its free-radical scavenging properties in extracellular compartments.

blood transport, as mechanisms for the probable regulation of intra-

Copper is well known as a biochemically essential transition element. Attribu-

COPPER IN ANCIENT MEDICINE

REDUCTION STATE OF RELEVANCE OF COPPER

O 2 (dioxygen) - dioxygen transport in

O 2-. (superoxide) - superoxide dismutation

H20 2 (hydrogen peroxide) - product of copper-

HO· (hydroxyl radical) - product of hydrogen

a constant release of low concentrations of pharmacologically reactive copper

addition, hydroxyl radicals produced as by-products in both copper-dependent

PARAMETERS USED TO EVALUATE THE BIOCHEMICAL ACTIVITY OF

SOD-active substances accelerate the decay of pulse-radiolytically generated

Factor V (proaccelerin) 1 x 330 1 Cu (type II) Blood clotting 157

Factor VIII 300 (80+220) 1 Cu (type I) Blood clotting 177

specific metal binding

human SA Asp ala - his - Iys - yes

of copper is found coordinated to albumin (serum concentration about 700

In order to elucidate whether or not a copper complex is stable under

*[N,N'-bis(2-pyridyl-methylene)-1 ,4-butanediamine]-(N,N' ,N",N"')-copper(II)

2R-SH+2Cu2 + -+ 2Cu+ +R-SS-R+2H+ (1)

Degradation of H 2 0 2 can be catalysed by copper compounds £10,11]. This

SELECTED COPPER COMPOUNDS

Table 2 Stability constants of Cu(II)-complexes

Ligand /OglO K/* References

Formate 1.57 0.65 7

*K. =[CuL]/[Cu] [L]; K2 =[CuLiJ/[Cu] [Ll'; L =ligand

questionable. Furthermore, copper complexes are designed as structural models

agents, such as the H 2 -receptor antagonists, cimetidine and famotidine, are

could modulate the action of NSAIDs by interfering with the distributing

ketoprofen and diclofenac have been designed in order to achieve a site-specific

BIOCHEMISTRY OF COPPER COMPOUNDS: AN EXAMPLE

*[N,N'-bis(2-pyridyl-phenyl-methylene)-1 ,4-butanediaminej-(N,N',N", N"')-copper(II)

activity of the complexes (measured under these conditions the superoxide

nitroxyl anion NO-2 + 102

NO + O~ ONOO- ---~ N03'

otherwise would decompose to nitrate via peroxinitrite (ONOO"), a very reactive

*[1 ,8-di(2-imidazoyl)-2, 7-diazooctadiene-l, 7]-(N, N',N", N"')-copper(JI)

Table 3 Superoxide dismutase activities of copper(U) complexes*

~~~~Ph": ]~t :=:O:d B-Hm:-,e-