dermatology - DermaAmin

DERMATOLOGY: CLINICAL & BASIC SCIENCE SERIES
COPPER and
the SKIN

DERMATOLOGY: CLINICAL & BASIC SCIENCE SERIES
Series Editor Howard I. Maibach, M.D.
Published Titles:
Bioengineering of the Skin: Cutaneous Blood Flow and Erythema
Enzo Berardesca, Peter Elsner, and Howard I. Maibach
Bioengineering of the Skin: Methods and Instrumentation
Enzo Berardesca, Peter Elsner, Klaus P. Wilhelm, and Howard I. Maibach
Bioengineering of the Skin: Skin Biomechanics
Peter Elsner, Enzo Berardesca, Klaus-P. Wilhelm, and Howard I. Maibach
Bioengineering of the Skin: Skin Surface, Imaging, and Analysis
Klaus P. Wilhelm, Peter Elsner, Enzo Berardesca, and Howard I. Maibach
Bioengineering of the Skin: Water and the Stratum Corneum,
Second Edition
Joachim W. Fluhr, Peter Elsner, Enzo Berardesca, and Howard I. Maibach
Contact Urticaria Syndrome
Smita Amin, Arto Lahti, and Howard I. Maibach
Copper and the Skin
Jurij J. Host´ynek and Howard I. Maibach
Cutaneous T-Cell Lymphoma: Mycosis Fungoides and Sezary Syndrome
Herschel S. Zackheim and Howard I. Maibach
Dermatologic Botany
Javier Avalos and Howard I. Maibach
Dermatologic Research Techniques
Howard I. Maibach
Dry Skin and Moisturizers: Chemistry and Function, Second Edition
Marie Lodén and Howard I. Maibach
The Epidermis in Wound Healing
David T. Rovee and Howard I. Maibach
Hand Eczema, Second Edition
Torkil Menné and Howard I. Maibach
Human Papillomavirus Infections in Dermatovenereology
Gerd Gross and Geo von Krogh
The Irritant Contact Dermatitis Syndrome
Pieter van der Valk, Pieter Coenrads, and Howard I. Maibach

Latex Intolerance: Basic Science, Epidemiology, and Clinical Management
Mahbub M. V. Chowdhry and Howard I. Maibach
Nickel and the Skin: Absorption, Immunology, Epidemiology,
and Metallurgy
Jurij J. Host´ynek and Howard I. Maibach
Pesticide Dermatoses
Homero Penagos, Michael O’Malley, and Howard I. Maibach
Protective Gloves for Occupational Use, Second Edition
Anders Boman, Tuula Estlander, Jan E. Wahlberg, and Howard I. Maibach
Sensitive Skin Syndrome
Enzo Berardesca, Joachim W. Fluhr, and Howard I. Maibach
Skin Cancer: Mechanisms and Human Relevance
Hasan Mukhtar
Skin Reactions to Drugs
Kirsti Kauppinen, Kristiina Alanko, Matti Hannuksela, and Howard I. Maibach

DERMATOLOGY: CLINICAL & BASIC SCIENCE SERIES
COPPER and
the SKIN
Edited by
Jurij J. Host´ynek
University of California at San Francisco School of Medicine
San Francisco, California, U.S.A.
Howard I. Maibach
University of California at San Francisco School of Medicine
San Francisco, California, U.S.A.
New York London

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This monograph on copper is dedicated to Dr. Roberto Milanino of the
University of Verona, Italy. A scientist and teacher, he spent the past 30 years
of his academic career investigating the biological function and importance of
copper in the mammalian organism while navigating the narrows of Italy’s
budget allocated to science. Among the leading authorities on this subject,
he was a guiding light, set the proper accents, and kept things in perspective
over the period of much of our work and writing in assembling this monograph.
The whole is a clear reflection of his qualities as teacher.
J. J. Hosty´nek
H. I. Maibach

Preface
Preface
Metals have long interested the dermatologic community in terms of their
toxicity and efficacy. A century ago, mercury enjoyed widespread usage in
the treatment of syphilis.
As the dermatologic sciences evolved, sufficient knowledge also evolved,
leading to the clear assertion that metals may be toxic when applied to the
skin. Nickel has enjoyed the greatest amount of study, especially because of
its frequent induction of clinical allergic contact dermatitis in humans. Cobalt
is also a common contact allergen, but its clinical significance is less clearly
explored. Chromate in cement, leather, and other applications also enjoys
considerable study. Most recently, gold salts have been recognized as a common
inducer of cell-mediated immunity; however, this clinical significance is
currently being investigated in terms of dermatitis and even restenosis.
These data have led to several textbooks dedicated to individual metals.
The first was on chromate (by Desmond Burrows), many metals and the skin
(by R. Guy and J. J. Hosty´nek), and most recently nickel (by J. J. Hosty´nek
and H. I. Maibach). The current volume on copper presents sufficient information
to place it among the pantheon of the metallic gods and dermatology.
Our aim was to mold contributions from individuals widely spread over
several disparate disciplines into a cohesive, readily digestible text. The
individual disciplines include basic chemistry (metallurgy), dermatotoxicology
(irritant and allergic contact dermatitis), and membrane transport
(percutaneous penetration).
v

vi Preface
Our specific objective is to allow scientists in many fields to more
efficiently focus their attention on this essential element (copper) and the
skin. Hopefully, the parts will simplify understanding the whole.
This volume differs from the others, not only in its extensive dermatotoxicologic
profile of copper and its salts, but also as an equally impressive
data on copper’s possible anti-inflammatory actions in man.
The editors welcome suggestions for the next edition.
Jurij J. Hosty´ nek
Howard I. Maibach

Acknowledgment
We gratefully acknowledge the partial financial support by the International
Copper Association, Ltd. (ICA) towards publication of this book.
vii

Preface . . . . v
Acknowledgment . . . . vii
Contributors . . . . xv
Contents
1. Copper and Copper Alloys
Harold T. Michels
. . . . . . . . ................ 1
Introduction . . . . 1
Copper: Properties of the Element . . . . 1
Pure Copper . . . . 2
Copper Alloys . . . . 2
Properties of Copper Alloys . . . . 2
Copper Alloy Families . . . . 4
The High Coppers . . . . 4
Conclusions . . . . 6
2. Corrosion Chemistry of Copper: Formation of Potentially
Skin-Diffusible Compounds . . . . . . . . ................
Jurij J. Hosty´nek
7
Introduction . . . . 7
Electron Configuration and Reactivity of Copper . . . . 8
Corrosion of Copper in the Environment . . . . 8
Corrosion of Copper in Physiologic Media . . . . 9
Conclusions . . . . 15
Glossary . . . . 16
Abbreviations . . . . 16
References . . . . 16
ix

x Contents
3. Basics of Metal Skin Penetration:
Scope and Limitations . . . . . . .................... 21
Jurij J. Hosty´ nek and Howard I. Maibach
Introduction . . . . 21
Structure of Skin and Its Function as
Diffusion Barrier . . . . 23
Descriptors of Dermal Absorption . . . . 25
Permeant Categories and Paths of Diffusion . . . . 28
Compounds Formed by Metals in Contact
with the Skin . . . . 31
Variables Determining Skin Diffusion
of Metal Compounds . . . . 35
Methods for Measuring Percutaneous Absorption . . . . 45
Analytical Methods for Metal Detection . . . . 53
Summary and Conclusions . . . . 56
Abbreviations . . . . 57
References . . . . 58
4. Percutaneous Absorption of Copper Compounds . . . . . . . . 67
Jurij J. Hosty´ nek and Howard I. Maibach
Introduction . . . . 67
Qualitative Diffusion Data . . . . 68
Semiquantitative Data . . . . 70
Quantitative Data . . . . 71
Discussion and Conclusions . . . . 73
Limitations in Measuring Copper Absorption
In Vivo . . . . 74
Interdependence of Systemic Copper and Zinc Levels . . . . 75
Recommendations for Research to Fill Existing
Data Gaps . . . . 76
Conclusions . . . . 77
Glossary . . . . 78
Abbreviations . . . . 78
References . . . . 79
5. Diffusion of Copper Through Human Skin
In Vivo . . .................................. 81
Jurij J. Hosty´ nek, Howard I. Maibach, and Frank Dreher
Introduction . . . . 81
Experimental . . . . 84
Results . . . . 85

Contents xi
Discussion . . . . 88
Conclusions . . . . 92
Glossary . . . . 93
Abbreviations . . . . 93
References . . . . 94
6. Irritation Potential of Copper Compounds . . . . ........ 97
Jurij J. Hosty´nek and Howard I. Maibach
Introduction . . . . 97
Exposure to Copper . . . . 97
Solubilization of Copper Metal . . . . 98
Incidence and Epidemiology of Irritation
Due to Copper . . . . 100
Pharmacology of Copper . . . . 101
Copper Irritancy in Skin and Mucosa . . . . 103
Conclusions . . . . 111
Abbreviations . . . . 112
References . . . . 112
7. Copper Hypersensitivity: Dermatologic
Aspects—Overview . . . ........................ 115
Jurij J. Hosty´nek and Howard I. Maibach
Introduction . . . . 115
Metallurgy of Copper and Its Alloys, and Its Role
as Sensitizer . . . . 117
Predictive Immunology Test Results
for Copper . . . . 119
Diagnostic Tests for Hypersensitivity . . . . 119
Test Concentrations for Copper ACD . . . . 123
Immunogenic Potential of Copper . . . . 123
Summaries of Population-Based Studies . . . . 134
Summary of Selected Case Reports of Immune Reactions
to Copper . . . . 138
Selection of Individual Reports of Immune Reactions
to Copper . . . . 138
Comments . . . . 140
Conclusions . . . . 140
Abbreviations . . . . 141
References . . . . 141

xii Contents
8. Copper in Medicine and Personal Care:
A Historical Overview . . . . . . ................... 149
Roberto Milanino
Introduction . . . . 149
The Sumeric Culture: Circa 4000–2300 B.C. .... 150
The Ancient Egyptian Culture . . . . 150
The Babylonian–Assyrian Culture:
Circa 1750–539 B.C. .... 152
The Ancient Indian Culture: Circa 2800–1000 B.C. .... 152
The Ancient Chinese Culture: Circa 3000 B.C.
to 1100 A.D. .... 152
The Pre-Columbian Meso- and South-American Cultures:
Circa 600 B.C. to 1500 A.D. .... 153
The Ancient Greek Culture . . . . 153
The Ancient Roman Culture: Circa 600 B.C.
to 476 A.D. .... 155
From the High-Medieval Age to the Early
20th Century . . . . 156
Beginning of the Scientific Age for Copper:
1928–1976 . . . . 157
Conclusions . . . . 158
Abbreviations . . . . 159
References . . . . 159
9. The Role of Copper in Onset, Development, and
Control of Acute and Chronic Inflammation . . . . . . . . . . 161
Roberto Milanino
Introduction . . . . 161
Studies on Copper-Deficient, Experimentally
Inflamed Animals . . . . 163
Laboratory Animals: Studies on ‘‘Endogenous’’ Copper
Metabolism in Acute and Chronic Inflammation . . . . 170
Human Subjects: Studies on ‘‘Endogenous’’ Copper Metabolism
in Acute and Chronic Inflammations, with a Particular
Reference to Rheumatoid Arthritis . . . . 179
Effects of ‘‘Exogenous’’ Copper Administration on the
Inflammatory Process . . . . 184
Copper Anti-inflammatory Activity: Hypotheses Explaining
the Possible Mechanisms of Action . . . . 203
Conclusions . . . . 216

Contents xiii
Abbreviations . . . . 219
References . . . . 220
10. Copper Jewelry and Arthritis . . . . . . .............. 237
Brenda J. Harrison
Introduction . . . . 237
The Copper Bracelet ‘‘Myth’’ and Hypothesis . . . . 239
The Copper Bracelet Trial . . . . 243
The Present State of the Copper Bracelets ‘‘Issue’’ . . . . 251
Is There Likely to Be a Future for Copper Bracelets
in Arthritis Care? . . . . 256
Appendix A: Position Statements of Support Organizations,
Government Agencies, Etc. . . . . 257
Appendix B: Miscellany . . . . 259
References . . . . 261
11. Role of Copper in Anti-inflammatory Therapy and the
Potential for Its Transdermal Application . . . . . ....... 267
Jurij J. Hosty´nek and Roberto Milanino
Introduction . . . . 267
Traditional and Modern Therapies for RA
and Related Disorders . . . . 268
Drug Therapy . . . . 271
Precedents in Topical Delivery of Anti-inflammatory
Agents . . . . 275
Role of Copper in AI Activity . . . . 275
Past Use of Copper Chelates in the Treatment
of Rheumatoid Arthritis . . . . 278
Transdermal Delivery of Anti-inflammatory Copper
Chelates vs. Conventional (Systemic)
Anti-inflammatory Therapy . . . . 278
Conclusions . . . . 286
Outlook . . . . 288
Abbreviations . . . . 288
References . . . . 289
Index . . . . 295

Contributors
Frank Dreher Neocutis, Inc., San Francisco, California, U.S.A.
Brenda J. Harrison Department of Earth and Ocean Sciences, Copper
Research Information Flow Project, University of British Columbia,
Vancouver, British Columbia, Canada
Jurij J. Hosty´nek Department of Dermatology, University of California
at San Francisco School of Medicine, San Francisco, California, U.S.A.
Howard I. Maibach Department of Dermatology, University of California
at San Francisco School of Medicine, San Francisco, California, U.S.A.
Harold T. Michels Copper Development Association, Inc., New York,
New York, U.S.A.
Roberto Milanino Facoltà di Medicina e Chirurgia, Sezione di
Farmacologia, Dipartimento di Medicina e Salute Pubblica, Università di
Verona, Verona, Italy
xv

1
Copper and Copper Alloys
Harold T. Michels
Copper Development Association, Inc., New York, New York, U.S.A.
INTRODUCTION
This first chapter describes copper, its properties and characteristics, and
where it is used, both in its pure form and as alloys. The emphasis is on
materials that come into contact with human skin. This chapter provides
the background for the second chapter, which gives a detailed discussion of
the corrosion resistance of these materials and how that relates to their
interaction with humans by sweat. Common items made of copper and
copper alloys that are touched by humans every day include copper and copper–nickel
coins, copper–nickel–zinc door keys, and brass door knobs, door
push plates, and sink faucet handles.
COPPER: PROPERTIES OF THE ELEMENT
Copper, atomic number 29, is classified as a metal in the periodic table of
elements. It is the first element in the group containing silver and gold,
and thus it is considered to be a semiprecious metal.
Some of the properties of copper include:
melting point of 1083 C
metallic luster and reddish color
high electrical and thermal conductivity
nonmagnetic
alloys readily as both a solute and a solvent
1

2 Michels
good corrosion resistance and durability
forms a protective oxide in air and water
face-centered crystal structure
high malleability, formability, and ductility
good machinability
readily electroplated
essential nutrient for life
highly recyclable
These unique properties of copper account for its widespread and
long-term use as an industrial material.
PURE COPPER
Copper is refined from ore shipped to fabricators, mainly as cathode, wire
rod, billet, cake (slab), or ingot. Through extrusion, drawing, rolling, forging,
melting, or atomization, fabricators form wire, rod, tube, sheet, plate,
strip, castings, powder, and other shapes. These copper and copper alloys
are then shipped to manufacturing plants where they are used to make
products to meet society’s needs.
COPPER ALLOYS
Copper alloys are widely used in many applications, ranging from electrical
wiring and connectors to musical instruments, from household plumbing
tube and fixtures to keys, locks, doorknobs, and handrails. The applications
are almost endless. The wide use of copper alloys is attributable to a long
history of successful use, ready availability from a multitude of sources, the
attainability of a wide range of physical and mechanical properties, and
amenability to subsequent processing, such as machining, brazing, soldering,
polishing, and plating. The properties of copper alloys, which occur
in unique combinations found in no other alloy system, include high
thermal and electrical conductivity, a wide range of attainable strength properties
and excellent ductility and toughness, as well as superior corrosion
resistance in many different environments.
Nevertheless, to the uninitiated, copper alloys appear to be confusing
and complex. They are generically described by such terms as brass, bronze,
copper–nickel, and copper–nickel–zincs, which are called nickel silvers
because of their shiny white color, even though they contain no silver.
PROPERTIES OF COPPER ALLOYS
Copper alloys provide important properties and characteristics including:
Good corrosion resistance—which contributes to durability, and
leads to long-term cost effectiveness.

Copper and Copper Alloys 3
Favorable mechanical properties—ranging from pure copper that is
soft and ductile to other alloys, such as the manganese bronzes that
can rival the mechanical properties of quenched and tempered
steel. Furthermore, almost all copper alloys retain their mechanical
properties, including impact toughness, at very low temperatures.
High thermal and electrical conductivity—copper has higher conductivity
than any other metal except silver. Conductivity drops
when copper is alloyed. However, even the copper alloys with relatively
low conductivity transfer both heat and electricity far better
than other corrosion-resistant materials, such as titanium, aluminum,
and stainless steel.
Biofouling resistance—copper inhibits the growth of marine organisms
including algae and barnacles. This property, unique to
copper, decreases when alloyed. However, it is retained at a useful
level in copper alloys, such as the copper–nickels, which are routinely
found in marine applications.
Antimicrobial action—copper chemicals have been historically used as
bactericides, algicides, and fungicides. However, recent studies indicate
that bacteria, including certain harmful strains of Escherichia
coli, and MRSA, or methicillin-resistant Staphylococcus aureus
(a serious nosocomial or hospital-acquired infection) simply die in a
few hours when placed on copper alloy surfaces at room temperature.
Low friction and wear rates—copper alloys, such as the high-leaded
tin bronzes, are cast into sleeve bearings and exhibit low wear rates
against steel. Both the nickel bronze and the tin bronze are the
industry standards for worm gears, an application in which low
wear rates are important.
Good castability—all are sand castable and almost all can be
centrifugally and continuously cast. Many copper alloys can be permanently
molded and precision or die cast. Wrought copper alloys
are initially cast and subsequently hot and cold rolled.
High fabricability—copper alloys are readily hot rolled extruded or
forged. They then may be cold rolled to the desired thickness.
Sheet, plate, strip, and bar products are readily forged, stamped,
and bent into desired shapes.
High machinability—good surface finish and high tolerance control
is readily achieved. While the leaded copper alloys are free-cutting
at high machining speeds, many unleaded alloys such as nickel–
aluminum bronze are readily machinable at recommended feeds
and speeds with proper tooling.
Ease of subsequent processing—many copper alloys are routinely
polished to a high luster, especially those with an esthetically pleasant
color, such as the yellow brasses. Plating, soldering, brazing,
and welding are also routinely performed.

4 Michels
Availability of a range of alloys—in a given application, any one of
several alloys may be a suitable candidate, depending on design
loads and corrosivity of the environment.
Reasonable cost—high processing yield and low machining costs
make copper alloy very economical. Gates and risers from castings
and chips from machining are also recycled, which leads to additional
overall cost reductions. In addition, copper alloys do not
require surface coatings, such as paints. The avoidance of surface
coatings further reduces initial costs and provides additional maintenance
savings. In addition, when the component reaches the end
of its useful life, it too is readily and routinely recycled.
COPPER ALLOY FAMILIES
From a metallurgical viewpoint, many copper alloys are single-phase solid
solutions in which the alloying elements, such as zinc, tin, and nickel, are
substituted for copper in the copper matrix. Examples of single-phase
solution alloys include the brasses that contain up to 35% zinc, and copper–
nickels that contain up to 33% nickel. As alloy content is increased, a second
phase may form. In the case of brass, when the zinc content is increased, a
hard second phase called beta forms within the alpha copper-rich matrix.
This second phase is found in yellow brass that contains up to 41% zinc.
Beta, which slightly impairs room temperature ductility, markedly increases
ductility at elevated temperatures. One of the most common systems used
to designate specific copper alloys is the UNS system. Copper alloys are
either wrought or cast. Wrought alloys range in UNS number from C10100
through C79999. They are subjected to hot and usually cold work after initial
melting and solidification, and are generally available as wire, rod, bar,
sheet, strip, and plate. Cast alloys range in UNS number from C80000
through C99999. They are typically cast into a mold in a variety of specific
shapes and then machined without any hot or cold working.
The Coppers
The coppers (wrought: C10100–C15999, and cast: C80000–C81399) are
essentially pure copper (99.7% min) with traces of silver or phosphorus.
The presence of silver imparts annealing resistance, while phosphorus, a deoxidizer,
aids in welding. These alloys, which have relatively low strength, are used
in applications where high thermal and electrical conductivity are desirable,
such as in electrical connectors and in hot metal handling. Copper is used
in low denomination coinage. Some individuals wear copper wrist bracelets.
THE HIGH COPPERS
High copper alloys (wrought: C16000–C19999, and cast: C81400–C83299),
which contain 95.1% copper (min), are unique in that they combine high

Copper and Copper Alloys 5
strength with high thermal and electrical conductivity, two properties that
are seldom found together in the same material.
Chromium coppers are alloys containing up to 1.2% chromium. The
strength of chromium copper is approximately twice that of pure copper,
but its electrical conductivity remains high (80% of pure copper). Applications
for chromium copper include welding clamps and high-strength
electrical connectors.
The Brasses
The brasses (wrought: C21000–C49999, and cast: C83300–C89999) are the
most commonly used casting alloys, and are essentially alloys of copper
and zinc.
The red brasses are alloys of up to 15% zinc and may contain varying
amounts of tin if they are cast alloys. Lead may be present in various
amounts to promote pressure tightness in castings in service and to facilitate
free machining during the manufacturing process. The color of red brass is
attributable to its relatively low zinc content. The largest-volume cast red
brass alloy, C83600 (commonly known as 85-5-5-5), contains 85% copper,
5% tin, 5% lead, and 5% zinc. It has been used commercially for several
hundred years and accounts for more tonnage than any other cast alloy.
The yellow brasses are even lower in cost than the red brasses because
their zinc content is higher, at 20–39% Zn. Yellow brass has a pleasant
yellow color, which can be polished to a high luster. This accounts in part
for its selection as decorative hardware.
The Bronzes
Bronze (wrought: C50000–C69999, and cast: C90000–C95999) is a very
imprecise term. Strictly speaking, it originally referred to alloys in which
tin was the major alloying element. Today, the term bronze applies to a
broader class of alloys, which may contain little, if any, tin.
The silicon bronzes and silicon brasses are essentially alloys of up to
20% zinc and up to 5% silicon. They have low melting points and high fluidity,
which favor permanent molding and pressure die casting.
Modern day cast tin bronzes are very similar to the alloys found in
many relics from the ‘‘Bronze Age,’’ over 3500 years ago. They are basically
alloys of copper and tin, where tin content can be up to 20%. The good
aqueous corrosion resistance of tin bronzes accounts, in part, for the survival
of these Bronze Age relics to this day. Additional attributes of tin
bronzes include reasonably high strength, good wear resistance, and a low
coefficient of friction versus steel, making them very useful for bearings,
piston rings, and gear parts.
Aluminum bronzes have complex metallurgical structures. Alloying
elements always include aluminum and varying amounts of manganese,

6 Michels
iron, and, in some versions, nickel. Aluminum imparts both strength and
oxidization resistance by virtue of the formation of alumina (Al2O3)-rich
protective films. These alloy are very wear resistant, and exhibit good casting
and welding characteristics. Their corrosion resistance is superior in
seawater, chloride, and in dilute acids. Applications are varied and include
propellers and valves, pickling hooks, pickling baskets, and wear rings. Aluminum
bronzes and especially nickel–aluminum bronzes are desirable alloys
for fluid moving applications such as pump impellers, because of superior
erosion-corrosion and cavitation resistance.
Copper Nickels
The copper–nickel alloys (wrought: C70000–C73499, and cast: C96000–
C96999) are simple solid solutions of nickel in copper. Nickel content varies
from 9% to 33%. A small amount of manganese (0.05–1.5%) and iron
(0.4–1.8%) is present. Their excellent corrosion resistance in seawater, combined
with their high strengths and good fabricability, accounts for their
wide use in piping, heat exchangers, valves, ship tail-shaft sleeves, and other
marine applications. They are also used in coins.
Nickel Silvers
Nickel silver alloys (wrought: C73500–C79999, and cast: C97000–C97999)
contain nickel (11–27%) and zinc (1–25%). The presence of nickel primarily
accounts for their pleasant silver luster but, in contrast to their name, the
nickel silvers do not contain silver. In spite of their high degree of alloying,
the nickel silvers are simple solid solution alloys. They offer good corrosion
resistance, ease of castability, and good machinability. Major uses include
hardware for food processing, seals, architectural trim, musical instrument
valves, and door keys.
Other Copper Alloys
In the interest of completeness, two other types of copper alloys, which are
both cast alloys, should be mentioned. They are the leaded coppers
(C89000–C98000), which are typically employed where their high lead provides
lubricity, and the special alloys (C99000–C99999), which have unique
specific properties needed for specialized applications.
CONCLUSIONS
The copper alloys offer a wide range of properties to meet the needs of many
applications that humans touch. The list is almost endless. End-use applications
are limited only by the knowledge, creativity, and imagination of those
who specify, design, produce, and process these copper alloy products.

2
Corrosion Chemistry of Copper:
Formation of Potentially
Skin-Diffusible Compounds
Jurij J. Hostynek
Department of Dermatology, University of California at San Francisco
School of Medicine, San Francisco, California, U.S.A.
INTRODUCTION
In the background of this review lies the question, what effect, if any, does
copper metal have when kept in contact with the skin, on inflammation, and
arthritis in particular—i.e., concerning the unsettled issue of the ‘‘copper
bangle.’’ Copper complexes given systemically have a well-documented therapeutic
effect on inflammatory conditions (1), and anti-inflammatory agents
(NSAIDs) have also proven successful by dermal delivery (2,3). Investigations
demonstrating the efficacy of dermal assimilation of copper in arthritic and
rheumatoid conditions by skin contact with the metal or its derivatives in
humans have been few, poorly conducted, and have only brought qualitative,
mostly subjective, evidence of benefits to be expected from the ‘‘mere’’ skin contact
with the metal (4). The review of the microenvironment prevailing on the
skin surface and the chemical agents present there aims to delineate acceptable
chemical arguments that support the contention that copper, solubilized in that
environment, is apt to be assimilated by the mammalian organism.
This chapter, in part, was reprinted from Hosty´nek JJ. Factors determining percutaneous metal
absorption. Food Chem Toxicol 2003; 41:327–345, with permission of Elsevier.
7

8 Hostynek
ELECTRON CONFIGURATION AND REACTIVITY OF COPPER
The transition metals in the periodic table have partially filled d (or f) electron
shells, in which an inner shell is being filled with electrons, two in each of five
orbitals, while the outer s-electron shell of slightly lower energy is complete.
Characteristic for transition metals is a low ionization potential (electropositivity),
i.e., the ease with which they can lose electrons to yield a variety of
positive ionic oxidation states, and the ability to form complexes. This occurs
mainly with sulfur, (e.g., in histidine), oxygen, or nitrogen groups (e.g., in
cysteine), particularly in biological systems and chelates (cyclic coordination
compounds) that are stable and likely to remain unchanged in physical
processes, such as membrane transport.
Copper has a single s electron outside the filled 3d shell, but the d electrons
also are involved in metallic bonding, and the (most common) Cu(II)
ion has the configuration 3d 9 , having lost the single s electron from the
outermost shell and one d electron. While further oxidation to Cu(III) is
difficult, the lower oxidation states can interchange easily. For instance,
the equilibrium 2Cu(I) D Cu(0)þ Cu(II) can be readily displaced in either
direction. Copper complexes with proteins—molecules composed entirely
of alpha-amino acid residues covalently united head to tail by peptide
bonds—to form unbranched polymers. Examples are copper metallothionein
(a small, ubiquitous protein in the organism fulfilling a multifunctional role
in absorption and reversible storage, transport and detoxification of the
highly toxic free copper ion) ceruloplasmin (the enzyme involved in the antiinflammatory
action of copper), blood clotting factors V and VIII, and
copper glycyl-L-histidyl-L-lysine, Cu(II)-GHK, which may prevent the inhibition
of mitochondrial glucose oxidation (5–8).
CORROSION OF COPPER IN THE ENVIRONMENT
For metals and their alloys exposed to the environment, the major reactions
are oxidation to the ionic form and liberation of electrons:
M ! M xþ þ x e
and the reduction of the (obligatory) oxygen present to form water:
O2 þ 4H þ þ 4e ! 2H2O
In presence of oxygen, on the surface of pure copper metal cuprous
oxide is formed, whereby copper ion persists in the monovalent Cu(I) state,
due to the reducing action of the metal. In the presence of saline (water),
oxygen dissolved in the medium can oxidize Cu(I) further to yield the cupric
ion, Cu(II), with formation of free radicals (9).
Although copper and its alloys have been known and widely used since
prehistoric times, appropriately named the Bronze Age, corrosion of the

Corrosion Chemistry of Copper 9
pure metal has hardly been investigated and is poorly documented. The corrosive
action of the environment (e.g., seawater) has focused on its alloys,
the bronzes and brasses, but not on pure metal itself (10). Well established
is the corrosive action of saline, a prime factor in the dissolution of the metal
(11), due to which copperware used in cooking is usually lined with tin.
While main sources of exposure to copper occurs through the diet (animal
organs, seafood, certain vegetables, and nuts), unsuspected exposure
may be due to release of copper corrosion products from copper cooking
and tableware (12), or the metal released into the plumbing system of water
conduits (13). Low hardness, low alkalinity, chlorinated tap water in municipal
water supplies, particularly of low pH and at elevated temperatures
(as in hot water conduits), increases the rate of corrosion. The effects of temperature,
chlorine, and organic matter on copper alloys have been investigated
in a number of studies (14).
Corrosion products of copper, brass, or bronze that are formed on
exposure to air, of different hues of blue and green, are collectively referred
to as verdigris, a term possibly derived from the term ‘‘vert de Grece,’’ mentioned
in ancient texts. A mixture of cupric acetate Cu(CH3COO)2, basic
cupric acetate Cu(OH) (CH3COO), and carbonate CuCO3, verdigris salts
occurring naturally are mostly noticeable as patina on weathering copper
objects, such as roofs and statues, described in Egyptian records dating
back to 1500 B.C. In post-Renaissance Europe, verdigris was used in oil-based
paint pigments to decorate interiors of homes, and as wood preservative.
Formed by the action of acetic acid vapors on metallic copper since the
Middle Ages and until the early 20th century, verdigris was produced in
the wine-growing regions of France as a cottage industry. Flat copper strips
were rubbed with verdigris, then layered with fermented grape husks for
several weeks until crystals formed on the metal surface. Dripping wine on
the surface promoted the formation of spongy green crystals that, scraped
from the copper, were dissolved in vinegar, recrystallized on wooden sticks,
crushed, and sold as pigment (15).
CORROSION OF COPPER IN PHYSIOLOGIC MEDIA
Copper and its alloys are present in numerous articles of everyday use, and
thus come in regular, sometimes extended and intimate contact with the skin,
or through systemic exposure by implanted prostheses or contraceptive
devices [intrauterine devices (IUDs)]. Heat, moisture, sweating, and friction
promote chemical reactions on the surface of objects in contact with the skin.
Skin: The Action of Sweat and Sebum
To gain insight onto the phenomenon of skin penetration by metals in
general, it is important to account for the factors involved in that process

10 Hostynek
stemming from the chemical microenvironment prevailing on the skin
surface, as the oxidative action encountered there is a determinant factor
in the formation of skin-diffusible derivatives. By the action of salts and
acids present in sweat and sebum, metals in their elemental state are converted
to the hydrophilic or lipophilic derivatives, respectively. Only as such
do metals become diffusible via the transcellular, intercellular, or transappendageal
route.
Leaching or release of copper ion from metal objects (alloys) in contact
with sweat is a multifactorial event that defeats prediction. Aside from
immediate environmental factors, the microenvironment within the particular
alloy in contact with a conducting medium (electrolyte, e.g., sweat) is a
principal determinant, due to the action of electromotive forces prevailing at
the interface of atoms with differing electromotive potential (16).
Such metals alloyed with copper form a galvanic element (or pile),
whereby an electron current flows from the more electronegative to the more
electropositive one, resulting in oxidation or solution (‘‘corrosion’’) of the
more electronegative metal. These effects, unpredictable quantitatively, vary
in function of actual metals present and their ratios in alloy composition.
A recent example with potential impact on public health is the recently minted,
bimetallic (cupronickel) 1- and 2-Euro coins, consisting of 25% nickel.
That bimetallic structure, which generates a potential difference of 30–40 mV
between the components, increases nickel release, due to galvanic corrosion,
facilitated in the medium of conducting sweat. As a consequence, reports of
nickel sensitizations from dermatology clinics are on the increase in a number
of countries where the new currency is in circulation, as nickel release in handling
the coins may elicit contact dermatitis in those allergic to nickel (17–22).
While amino acids, such as glycine or histidine, or proteins that can
complex with cupric ions enhance the rate of copper released, the presence
of saline and oxygen are prime factors in the dissolution of the metal. In
their absence no copper is released (9,23).
As formulated for the general reaction of a metal with amino acids or
fatty acids present in sweat, the process can be presented as:
2M 0 þ O2 þ 4HL ! 2ML2 þ 2H2O
where M 0 is the metal in elemental form and HL the endogenous ligand.
The Major Sweat Components
Electrolytes
The considerable variation in electrolyte composition occurring naturally in
sweat became evident in a number of studies conducted on the subject.
In one study on normal subjects, in Palmar sweat both sodium and
chloride levels fell below 50 milliequivalents/L (mEq/L) for 99% of subjects,
and below 65 mEq/L in all 649 volunteers tested (24).

Corrosion Chemistry of Copper 11
Using direct reading ion-selective electrodes, mean sodium concentration
in forearm transudate was 1.7 mEq/L 0.7, and chloride concentration
2.8 mEq/L 3.5 (n ¼ 6) (25).
Mean body sweat induced through exercise contained 24.2 2.2 mEq/L
chloride ion in men, and 26.0 4.6 in women (26).
Sodium and potassium content of pharmacologically stimulated sweat
in men (pilocarpine, methylcholine, and acetylcholine) was seen to be higher
than that measured in thermal sweat: mean values (in mEq/L) are 8.3 0.66
and 4.9 0.17, respectively (27).
Sweat osmolality values in normal adults were seen to increase with
increasing age. Range/mean (SD) values for men are 49–151/117 mmol/kg
(33.4) and for women 66–187/134 mmol/kg (38.6). An increase in osmolality
was observed to increase in tandem with the (normal) increase in
sodium concentration in sweat among an aging population (28).
The main cause for corrosion of metal surfaces from skin contact in
individuals referred to as ‘‘rusters’’ is not due to elevated electrolyte concentration,
as generally assumed, but rather seems to coincide with palmar
hyperhydrosis. When the sodium concentration measured in normal subjects
was compared to that of ‘‘rusters,’’ in fact no significant difference
could be observed (mean values of 49.6 vs. 49.1 mEq/L, respectively) (29).
Amino Acids
Proteins and AAs are normal components of mammalian sweat. The data
documented for humans differ significantly, probably due to differences
in the stimulation methods applied, to regional differences in anatomic site,
or the sampling methods used. Substantial variations were observed in relative
concentrations of AAs in sweat collected from various areas of the
skin, and their concentrations increased markedly in blood and urine on
oral protein intake (30). No differences in AA patterns were seen between
young and middle-aged adults, or between men and women (31). In
contrast to essential elements, AAs are neither selectively excreted nor
reabsorbed (32).
Large individual differences occur in AA composition between eccrine
forearm sweat from men under controlled exercise conditions. Comparison
of AA excretions analyzed in sweat and urine by ion exchange chromatography
showed comparable losses in those two media. In general, AA
concentrations were considerably higher in the exercise sweat of untrained
men than in the sweat of trained men determined by thermal and physiological
stimulation. Total average AA values collected from 20 trained
men and 20 untrained men were 12,797 and 24,855 mmol/L, respectively.
For trained versus untrained men, highest values were seen for serine
(3954/7782), glycine (2239/4392), alanine (1556/3028) and threonine
(1057/1856), respectively (33).

12 Hostynek
Lactate and Pyruvate
Lactate is the major organic compound secreted in sweat. At rest and low
sweating rates, the range in lactate concentrations is 30–40 mmol/L, at
higher sweat rate levels 10–15 mmol/L. Under thermal stimulation and also
during exercise, lactate concentration in sweat decreases with increasing
sweating rate, but remains constant in blood. Differences in the excretion
of lactate were also observed in function of physical fitness of three male
volunteers: mean values for the sedentary individual was 21.71 0.85, for
the fit individual 16.75 0.99, and the very fit individual was 12.75
0.50 mmol, respectively (34).
In contrast, pyruvic acid (pyruvate) concentrations in sweat are low,
found to vary between 0.1 and 1.2 mmol/L. The ratio of the two metabolites,
lactate and pyruvate, was observed to increase with rising heart rate (35).
Sebum
Human skin features an acid mantle of pH 4–6 at the surface of the SC in
normal, healthy subjects, which increases with depth to pH 7 at the juncture
with live tissue (36). Determinants of this pH are protons originating in the
epidermis or as products of sebaceous gland activity, which gradually reach
the surface of the skin. They stem from three classes of compounds:
amino acids (e.g., urocanic acid, pyrrolidone carboxylic acid)
alpha-hydroxy acids (e.g., lactic and butyric acid, also present in
sweat)
acidic lipids (e.g., cholesteryl sulfate and free fatty acids, primarily
oleic, linoleic, and behenic) (37–40)
Sebum as secreted by the sebaceous glands is a complex mixture of
lipids consisting of glycerides, but no free fatty acids. The occurrence
of free acids in the SC and on the skin surface is the result of hydrolysis of
phospholipids and glycerides by lipolytic enzymes occurring in the sebaceous
ducts and on the skin surface, and of bacterial decomposition. On
the skin surface, lipids of epidermal origin contain up to 20% free fatty
acids, those originating in the pilosebaceous glands, 16% (40). They consist
for the greater part of C16 and C18 acids, but their full range reaches
from C5 to C22, with an average length of C16 (39,41). Such an acid
milieu plays a regulating role for SC homeostasis, with relevance to the
integrity of the skin’s barrier function and regeneration of the SC (42).
It is believed that the acid environment on the skin surface both controls
moisture loss from the epidermis and protects the skin from fungal and
bacterial infection.
These acid components making up the sebum also play an important
role in solubilizing (‘‘corroding’’) metal surfaces in measurable amounts.

Corrosion Chemistry of Copper 13
The Release of Copper Ion in Artificial Sweat
The definition and thus reconstitution of human sweat for experimental purposes
fluctuate in function of numerous factors, which is why experimental
results are not consistent. Most striking are changes in sweat composition,
due to the rate of sweat secretion. Sodium and chloride content, one decisive
factor in the corrosion of copper and metals in general, is as low as 5 mEq/L
under quiescent conditions, due to a reabsorption (conservation) mechanism
(43,44). As sweating rate increases, that control mechanism is overwhelmed
and the sodium concentration can rise to approximate that occurring in
plasma. Also other significant components of sweat such as urea, lactic acid,
and potassium ions, increase at high rates of secretion and their concentrations
can also reach the levels of plasma.
Release of copper ions from various copper alloys in synthetic sweat
was investigated in model experiments.
Over a period of 24 hours at 35 C and a pH of 5.1, the range of Cu 2þ
liberated in artificial sweat was 80–100 mg/mL sweat (45). Walker and
Keats performed a number of experiments to determine the solubility of
copper metal in artificial sweat. In samples where initial copper concentration
was of the order of 2 10 5 M, after 24 hours the samples turned blue
and the concentration had increased to 2 10 3 mol Cu (46).
Oxidation of Copper in Contact with the Skin
Walker and Keats also measured the loss from copper bracelets worn by
volunteers. Bracelets measuring 22 1.3 0.1 cm lost 80 mg in 50 days when
worn around the ankles, and 90 mg when worn around the wrist (4).
Weight losses of 0.1% to 0.8% were observed for copper bracelets
weighing 14 g used in a trial involving volunteers and lasting one month
(47). Such variation can be explained by differential mechanical wear, but
a more likely alternative explanation is the variability in subjective sweat
composition and sweating rate.
Skin Penetration Data
Although limited in number, experiments have demonstrated that copper
compounds will penetrate the integument in humans and animals, mostly
in a qualitative manner. Such information is based on clinical observations
of remedial action in inflammatory diseases, describing therapeutic effects,
and qualitative observation by spectroscopic methods (Warner RR, 1993,
personal communication) (48–51).
One experiment only measured diffusivity of a skin-identical complex,
bis(glycinato) copper(II), in vitro on cat skin (52). A radioactive ( 64 Cu)
0.05 M solution in physiological saline applied to excised cat skin revealed,

14 Hostynek
after a lag time of nine hours, a steady-state transport rate described by a
Kp¼ 24 10 4 cm/hr.
Electron micrographs of skin sections stained for copper revealed the
metal in all layers of the skin in exposed samples. In 24 hours, 3.3% of
the applied copper(II) complex had completely penetrated the skin. This is
equivalent to 3.0 mg of the complex. Atomic absorption analysis found
47 ppm copper in the washing solution beneath the skin (20 mL isotonic
saline). This corresponds to 0.94 mg, which, in agreement with the radioactivity,
is equivalent to 3.1 mg of the complex.
To examine whether skin exudates will oxidize the surface of copper
metal in contact with skin, forming potentially diffusible compounds, copper
powder was applied under occlusion on the volar forearm of human
volunteers at UCSF. When the application areas were sequentially stripped
with adhesive tape and the strips then analyzed for metal content by inductively
coupled plasma-mass spectroscopy, a concentration gradient became
evident from the skin surface to the subcutis, characteristic of a passive diffusion
process. The results indicate that in contact with skin compounds are
formed, which can penetrate the intact stratum corneum to the level of live
epidermis (unpublished study).
Release of Copper in the Human Organism and
Its Contraceptive Effects
In the presence of saline, amino acids (e.g., glycine or histidine) or proteins
that complex with cupric ions accelerate the release of copper by removing free
copper ions from equilibrium. Copper incubated in a saline medium will cleave
the S–S bond, and serum albumin will undergo a conformational change (11).
Incubation with copper metal was seen to be spermatocidal at cupric ion concentrations
below 10 3 mol (53), and enzymes important in the implantation
process were also inactivated on incubation in the presence of copper metal
in a saline medium. Copper metal incubated in saline in the presence of oxygen
produces free radicals (9), and it appears probable that free radicals thus
formed are responsible for the contraceptive action of copper in IUDs.
The ‘‘copper IUD,’’ a plastic T-shaped device with copper wire wound
around its stem, has an increased contraceptive effect in women as compared
to the T insert without copper, and it continues to be a popular method of
temporary contraception. With the aim of obtaining insight into the mode
of action of these devices, studies were conducted to investigate the effect of
copper IUDs on the uterine milieu in women. The chemical process involved
on the surface of the metal in the uterus appears responsible for the contraceptive
activity (i.e., inhibition of implantation) (54).
In solutions of saline and serum albumin, a strip of 200 mm 2 copper
dissolved at a rate comparable to that of copper IUDs when measured
in vivo (23). Such losses of copper metal were determined on a number of

Corrosion Chemistry of Copper 15
copper IUDs in the actual use environment, by weighing before their insertion
into the uterus and after their removal a year later. Following one
year’s use, the loss from two sets of IUDs (120 and 135 mm 2 surface area)
amounted to 10.0 2.2 and 11.0 2.8 mg, respectively, the average release
per day being calculated at 28.7 mg (55).
Increases in systemic copper levels via parenteral exposure from a copper
IUD can also lead to adverse effects, even though the amounts liberated
from such a device are relatively low. Frentz and Teilum (54) traced induction
of copper hypersensitivity in patients to the action of a copper IUD.
They measured copper released from the device at 90 mg/day.
A fertility-related phenomenon due to the local increase in systemic
copper ion was described by Vesce et al., who noted that the use of the copper
intrauterine device was effective in the management of secondary amenorrhea.
In 40 of 48 volunteers with functional secondary amenorrhea, regular
menses were restored after insertion of an IUD, and normal menses were
maintained as long as the IUD remained in place. After removal of the
device, the effect persisted for one year. The authors of that study ascribe
the mechanism of action to the copper ion-mediated release of prostaglandins
from the endometrium (56).
CONCLUSIONS
Copper and its alloys are subject to chemical reactions on exposure to
environmental or physiological factors, whereby products are potentially
generated, which become diffusible through mammalian skin. The chemistry
of oxidation is reviewed as well as the factors contributing to corrosion. Skin
exudates (sweat and sebum) can react with metal surfaces that they come in
contact with, but even in the healthy organism their composition is variable,
in function of physical, pharmacological and environmental conditions,
gender, age, sweat rate, or body site. This overview addresses sweat and
sebum composition, and discusses components that determine the skin’s
corrosive action: chloride ion, low molecular weight acids and amino acids
(AAs) in sweat, and fatty acids in sebum, which hold the potential to solubilize
copper-containing metal objects. These components can form copper
salts and soaps whose molecular characteristics (size and polarity) will
determine rate and route of cutaneous penetration.
Rate and degree of copper corrosion is subject to a number of variable
environmental and biological factors that make predictions unlikely. Oxygen
and saline solutions are the main corrosive factors.
Elemental copper in the mammalian organism is highly reactive and
the resulting ions have biological effects.
In contact with the skin, metallic copper is measurably oxidized by
exudates under formation of derivatives that, at least in animals, penetrate
the integument as measured by the resulting anti-inflammatory activity.

16 Hostynek
In the living organism, corrosion becomes manifest through biological
evidence of contraceptive and spermatocidal action, resulting from the oxidation
process and products of copper IUDs. Conversely, evidence was presented
that in amenorrheic women the copper IUD can restore regular menses.
GLOSSARY
Corrosion Electrochemical process involving movement of
electrons, through a metal from anodic to
cathodic areas, and corresponding movement of
ions in the electrolyte medium.
Electromotive force Difference in driving force toward electron loss
between two metals causing a flow of current,
expressed in volt.
Ionization potential Energy (electron volts, ev) required to remove an
electron from its atomic orbit, with the value for
the standard hydrogen electrode set at 0.00 ev as
an arbitrarily selected standard.
Metallic elements Characterized by luster, malleability, conductivity
(thermal and electrical), and ability to form
positive ions.
Oxidation Process of electron removal from an atom or ion,
or the increase in the proportion of oxygen in
a compound.
Oxidation potential Electrical driving force toward electron loss,
expressed as a potential value (in electron
volts, ev).
Penetration Process of an exogenous agent entering one
skin layer.
ABBREVIATIONS
AA amino acids
L ligand
mEq/L milliequivalents per liter
SC stratum corneum
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a saline solution of bis(glycinato) copper(II). Bioinorg Chem 1977; 7:271–276.
53. Jecht EW, Bernstein GS. The influence of copper on the motility of human spermatozoa.
Contraception 1973; 7:381–401.
54. Frentz G, Teilum D. Cutaneous eruptions and intrauterine contraceptive
copper device. Acta Derm Venereol (Stockh) 1980; 60:69–71.
55. Hagenfeldt K. Studies on the mode of action of the copper-T device. Acta Endocrinol
Suppl (Copenh) 1972; 169:3–37.
56. Vesce F, Jorizzo G, Bianciotto A, Gotti G. Use of the copper intrauterine device
in the management of secondary amenorrhea. Fertil Steril 2000; 73:
162–165.

3
Basics of Metal Skin Penetration:
Scope and Limitations
Jurij J. Hosty´nek and Howard I. Maibach
Department of Dermatology, University of California at San Francisco
School of Medicine, San Francisco, California, U.S.A.
INTRODUCTION
The skin as target organ presents imponderable and wide margins of variability.
In vivo, permeability is subject to homeostasis regulating the overall
organism; in vitro, the sections of skin used for diffusion experiments are
likely to present artifacts. To further complicate the matter, diffusion of
metals appears to defy laws empirically derived for passive diffusion across
biological membranes. Endeavors to define rules governing skin penetration
by metals toward derivation of predictive quantitative structure–diffusion
relationships for risk assessment, thus, have been unsuccessful, and penetration
of the skin still needs to be determined separately for each metal compound,
by in vitro or in vivo assays. Because diffusion through biological membranes
is highly metal specific, and, in addition, metal ions’ valence is mutable during
the process of diffusion, molecular physicochemical parameters alone do not
suffice to model migration of electrolytes into and through the strata of the
skin. Certain factors are closely interrelated, and their combined effects are
neither entirely understood nor predictable. For example, unless the dynamics
This chapter, in part, was reprinted from Hosty´nek JJ, Maibach HI. Skin penetration by metal
compounds with special reference to copper. Tox Mech Meth 2006; 16:245–265, with permission
of Informa Healthcare.
21

22 Hosty´nek and Maibach
of in situ changes in speciation (oxidation state), or electrophilic reactivity,
among others, can be factored in, metal diffusivity will elude modeling.
Experimental data available so far from in vitro and in vivo experimentation
have been acquired under disparate conditions, and the base is too thin to
allow development of a predictive algorithm considering the number of metals
and metalloids of variable valence existing as free ions or forming chelates,
coordination compounds, or complexes with electron donors, such as oxygen,
sulfur, or phosphorus active in biological systems. The number of metals
discussed in this review may appear limited; this is due to the circumstance
that most scientists and government agencies so far have given priority to
the limited number of industrial materials, which comport special risks from
work place exposure in their investigations.
Healthy and intact skin was formerly considered to be an impermeable
barrier designed to shield the living organism from environmental
injury, also affording protection from chemical agents. More recently,
however, the skin is being recognized as a membrane, which can act as
filter, impenetrable to microorganisms but selectively pervious to chemicals
within limits of size and polarity, allowing molecular passage in as
well as out of the skin, and acting as a permanent depot or as dynamic
reservoir for essential trace elements (ETEs) and xenobiotics. Only larger
structures (e.g., microorganisms, proteins, and polymers) or highly polar
compounds, such as sugars, will not diffuse measurably through that barrier.
Although exposure to exogenous agents due to skin absorption is
usually less than through inhalation or ingestion, the large surface area
of the body is now recognized as a pharmacologically relevant port of
entry for both nutrients and xenobiotics. Dermal absorption can present
substantial risks in case of exposure to toxins, be they of natural or of
anthropogenic origin. Dermal exposure presenting the potential for
percutaneous absorption has become an integral part of toxicological risk
assessment, especially after incidents of significant morbidity and mortality
following exposure to agents of facile skin diffusivity. Thus, 36 infants
died in France upon exposure to baby talcum powder erroneously formulated
with a high dose of the neurotoxic antimicrobial hexachlorophene
(1–3) or, more recently, to dimethyl mercury, which proved lethal upon
skin contact in trace amounts (4).
Conversely, the skin is also an important secretory organ, which plays
a vital part in the process of detoxification and the maintenance of homeostatic
balance. By that token, on the one hand it is an important factor in the
organism’s clearing mechanism, because it allows elimination of toxics, such
as certain heavy metals, but, on the other hand, it can also lead to significant
elimination of ETEs, such as the detrimental loss of copper through excessive
sweating due to elevated temperature or physical strain. Excretion levels
of six trace metals examined in exercise-induced sweat placed copper in first
place (5,6).

Basics of Metal Skin Penetration: Scope and Limitations 23
Assessment of cutaneous absorption of metal compounds by the use of
new, refined analytical tools is reviewed here. This route of absorption may
be of pharmacological interest in order to compensate for shortages of
ETEs, due to nutritionally caused shortages or genetically caused defects
in intestinal absorption of metals, such as copper. Such shortages in nutrients
may be corrected through cutaneous dosing, as could be shown in
the case of zinc in humans (7–9) and in the rat (10).
It is the objective of this review to identify and describe the numerous
factors that need to be weighed critically when evaluating in vivo or in vitro
data stemming from various sources, and also in planning new such experiments.
Here the term ‘‘metal’’ refers to metals in all its forms of occurrence:
metallic state, electrolytic (ionized) form, and organometallics, complexes,
coordination compounds, or chelates. The term absorption is used to describe
metal penetration of one or several skin strata; penetration signifies metal
becoming available systemically.
This review addresses skin physiology and the various aspects of metals’
skin penetration according to the current scientific knowledge. This includes
discussion of:
1. Structure of the skin and its function as diffusion barrier
2. Descriptors of dermal absorption
3. Permeant categories and paths for their diffusion
4. Metals derivatives formed in contact with skin
5. Variables in skin diffusion by metal compounds
6. Methods for measuring skin permeation
7. Analytical methods for metal detection
STRUCTURE OF SKIN AND ITS FUNCTION
AS DIFFUSION BARRIER
Skin is a major organ involved in the maintenance of the body’s homeostasis:
its composition, heat regulation, blood pressure control, and excretory functions.
It is a multilayered organ, which is a living envelope consisting of
dermis and epidermis, with the stratum corneum (SC) as the outermost layer.
In men, the latter is a 10- to 40-mm thick layer of keratinized epithelial cells,
variable by anatomical site, held together in a ‘‘tongue-and-groove’’ arrangement.
It represents the rate-limiting diffusion barrier for most chemical
compounds (11,12), particularly limiting the loss of water (13). SC thickness,
seemingly a simple measurement, remains at best an estimate. Schwindt
et al. summarize previous efforts, adding indirect measurements in vivo in
men, utilizing transepidermal water loss (TEWL) measurements based on
Kalia’s diffusion equation (14,15). The SC is composed of layers of proteinaceous
cells separated by layers of lipid material. In humans it consists of
about 40% protein, 15% to 20% lipids, and 40% water (16). Likened to a

24 Hosty´nek and Maibach
brick-and-mortar structure, the SC consists of corneocytes (bricks) embedded
in an intercellular matrix of lipids (mortar), rendering the membrane poorly
permeable to water and other polar compounds (17). Interspersed in this
envelope are appendageal shunts, openings for hair follicles, and sweat
ducts. Underlying the SC are the strata of the avascular viable epidermis,
consisting of keratinocytes, the source of corneocytes. The epidermis is also
transversed by shunts: the hair follicles and sebaceous and sweat glands. In
ascending order, the human epidermis consists of the stratum basale,
responsible for the maintenance of epidermal–dermal adhesion. As cells
move outward from the basement membrane, in the process of differentiation
they undergo keratinization, transition to the stratum spinosum, then
further to the stratum granulosum. The latter consists of electron-dense
keratohyalin granules, which are irregularly shaped, nonmembrane-bounded,
electron-dense granules that contain profilaggrin, a structural protein, and a
precursor of filaggrin that plays a role in keratinization and barrier function.
The uppermost layers of the stratum spinosum and stratum granulosum
contain small membrane-bounded organelles known as lamellar granules,
Odland bodies, lamellated bodies, or membrane-coating granules. These
are the granules that contain lipids that can fuse to the cell membrane to
release its lipid content that will fill the intercellular space (18). The stratum
granulosum is also source of corneocytes, which, keratinized, form the SC,
the outermost layer of dead cells constituting an efficient barrier against
transcutaneous water loss. Besides generating the SC, other important functions
of the epidermis are metabolism of xenobiotics, synthesis of melanin by
melanocytes, and provision of a first-line immune defense by means of dendritic
Langerhans’ cells. In its entirety, the epidermis with the intercellular
lipids between the SC layers constitutes the rate-limiting barrier to the
absorption of both hydrophilic and lipophilic xenobiotics. Transition from
the lipophilic SC to the hydrophilic medium of the epidermis constitutes a
discontinuity that forms an important second barrier, largely inhibiting
further passage of lipophilic compounds once they have transited through
the SC. In its entirety, from skin surface to circulatory system the epidermis
consists of aqueous and lipid barriers. Removal of the hydrophilic epidermal
layer in in vitro diffusion experiments enhanced the percutaneous absorption
of lipophilic compounds across the SC, because it no longer functions as that
rate-limiting secondary barrier for the diffusion of lipophilic compounds
across the skin (19).
The support system for the epidermis is the underlying dermis, carrier
of nutrition through its vascular network; it is also involved in the immune
function through the lymphatic system, macrophages, and the mast cells
responsible for immune and inflammatory responses. It produces elastic
connective tissue consisting of collagen, laminin, and fibronectin, among
other components, and is the origin of sebaceous glands, eccrine and apocrine
sweat glands, and hair follicles.

Basics of Metal Skin Penetration: Scope and Limitations 25
DESCRIPTORS OF DERMAL ABSORPTION
Mostly, but not exclusively, description of dermal absorption is based on
two approaches:
The permeability coefficient, Kp, usually obtained through in vitro
experiments for compounds from water where it can be measured
directly
Percent absorbed as fraction of dose applied in vivo
The Permeability Coefficient K p
The permeability coefficient Kp—a flux value, normalized for concentration—
is a widely applied benchmark, representing the rate at which chemicals
penetrate the skin. It is derived by Fick’s law of diffusion (20) from flux measurements
through a biological barrier under conditions of steady state (J ss)
from an infinite reservoir of the compound, through a biological barrier,
and normalized for the concentration (C) applied: Jss ¼ Kp DC, e.g.,
expressed as cm/hr. Formulated to characterize passive diffusion of compounds
across membranes in general, Fick’s law also applies to passive
diffusion of xenobiotics through the SC of the skin. The law states that
if the permeation process reaches the point of steady-state equilibrium (i.e.,
the concentrations in the donor and receptor phases remain constant over
time), the steady-state penetration flux J ss per unit path length (here in cm)
is proportional to the concentration gradient dC and to the penetrant’s
permeation constant.
In practice, in a typical experiment the donor solution is scaled in such
a fashion that over the term of the experiment the concentration remains
‘‘practically’’ constant (a.k.a. ‘‘infinite’’ dose); because the receptor phase
is constantly removed, receptor concentration equals zero. Thereby, the
expression DC becomes equal to C, the concentration in the donor solution,
and the equation is simplified as
Jss ¼ Kp C or Kp ¼ Jss=C
The technique, easily standardized, allows the determination of Kp
through skin or other membranes as long as the barrier properties are not
affected by either permeant or carrier solvents. This implies that the permeant
may not react with the barrier material (i.e., change barrier permeability with
time). Because several heavy metals react with epidermal protein to some
degree, however this somewhat prejudices the general validity of K p values
(see section on variables).
Expressing depth of diffusion per unit of time, the Kp provides a basis for
comparison of the relative absorption rates of diverse chemicals. When the
permeant is applied from an aqueous solution, as is the case for most metal

26 Hosty´nek and Maibach
compounds, it also represents the method by which most compound-specific
penetration data currently available have been generated, for drugs as well as
hazardous pollutants (21,22).
The Kp, here expressed in cm/hr, is the most convenient parameter for
comparison of percutaneous penetration for purposes of dermatopharmacokinetics
and dermatotoxicology. Values for metal derivatives range from
7 10 7 cm/hr for nickel dichloride (23) to 2.1 10 3 cm/hr for sodium
dichromate (24) through human skin.
Discrepancies in Kp values reported by different investigators often
depend on whether mass balance was part of the study (i.e., whether the results
included material remaining in the barrier material at the end of the study).
For both dermatotoxicological and dermatopharmacological purposes, such
materials should be considered part of overall absorption unless it is
ascertained that the permeant does not diffuse further to reach the systemic
circulation. Transition metals especially tend to react with SC protein and
are retained in the outer strata, sometime to a degree significantly exceeding
amounts reaching the receptor phase.
Percent of Dose Absorbed
Relevant for purposes of risk assessment is the total amount of chemical systemically
absorbed. By that token it appears more important to determine
the amount which has disappeared from the site of application (surface
of the skin) and which eventually will flux through the skin and into the
organism and not the amount which is collected in the receptor phase in vitro
over the (necessarily) limited amount of time of the experiment. It thus
appears important that, whenever possible, absorption data be determined
in vivo, in men or primates, for as accurate a risk assessment as possible,
avoiding the uncertainties of cadaver skin or adjustments for species variation.
One principal in vivo methodology, using human volunteers or monkeys, is
the one developed by Feldmann and Maibach (25–27) and is the most applied
in vivo method for the determination of skin absorption, yielding much of
the data so far available on skin penetration by drugs and pesticides.
In human studies, following topical application, plasma levels of test
compounds are low, often falling below assay detection, and so it becomes
necessary to use radiolabeled chemicals. The compound labeled with
carbon-14 or tritium, or a metal isotope, is applied to the skin in a minimal
volume of a volatile solvent that is left to evaporate, and the total amount of
radioactivity (RA) excreted is then determined. The amount retained in the
body is corrected for by determining the amount of RA excreted following
parenteral administration. The resulting RA value is then expressed as the
percent of the applied dose absorbed. Fick’s postulates for membrane diffusion
are not met there because a concentration of the penetrant cannot be
defined and neither is a steady-state equilibrium reached with this method;

Basics of Metal Skin Penetration: Scope and Limitations 27
thus a permeability coefficient characterizing the compound can only be
calculated through prior conversion as given below, yielding an ‘‘apparent’’
Kp. When diffusion experiments are reported as the percent of the applied
dose, disappearance over a time interval t is given either as [% lost/(time)],
or as a first-order disappearance constant, k (min 1 ), for that time interval.
Assuming steady-state conditions, either of these parameters can then be
converted to the disappearance over a time interval t and is given either
as [% lost/(time)], or as a first-order disappearance constant, k (min 1 ),
for that time interval. Assuming steady-state conditions, either of these
parameters can then be converted to the permeability coefficient, Kp. That is
Kp ¼ J ð% lossÞ
¼
DC ½timeðhrÞŠ
CA VA
¼
100 A CA
ð% lossÞ
½timeŠ
VA
100 A
where J (mol/cm 2 /hr) is the chemical flux, C (mol/L) is the chemical concentration
gradient across the skin (which is reasonably assumed to be equal
to the applied concentration C A), V A (mL) is the volume of chemical solution
applied, and A (cm 2 ) is the area of application.
It follows, therefore, that, for a five-hour application of 1 mL of solution
to a 3.14 cm 2 area of skin
Kp;5 ¼ ð% lossÞ 5
1570
Kp ¼ J ðfraction lostÞ
¼
DC ½timeðhrÞŠ
CA VA
A CA
where, assuming first-order disappearance kinetics (characterized by rate
constant, k, min 1 ),
ðfraction lostÞ ¼1 expf 60 k ½timeðhrÞŠg
Thus, for a one-hour application of 1 mL of solution to 3.14 cm 2 of
skin, Kp is calculated as
Kp ¼ð1 expð 60 kÞÞ=3:14
Together with the toxicity of a chemical the percent of a substance
absorbed is the second most critical factor for risk assessment; it gives a measure
for human exposure and an indication of the amount potentially
absorbed in (the worst) case of total body immersion. For a chemical applied
at 100 mg/cm 2 , at 1% absorption, for instance, the extent of systemic exposure
to the compound applied on the entire body area of an average human
adult (18,000 cm 2 ) would be in the milligram range in the course of 24 hours.
ð1Þ
ð2Þ

28 Hosty´nek and Maibach
PERMEANT CATEGORIES AND PATHS OF DIFFUSION
Several routes are available for the diffusion of compounds into and through
the matrix of the skin. In order to facilitate a closer discussion of the alternatives,
it is useful to associate them with the different categories of
permeants as they transit alternate domains of the skin.
Organic Compounds (Nonelectrolytes)
The lipid matrix, which cements the corneocytes of the SC, is believed to be
the principal route of transport for nonelectrolytes and lipophilic (organic)
compounds into the deeper layers of the integument. Most extensively investigated
so far is the process of intercellular diffusion by compounds from the
categories of drugs, pesticides, organometals, and cosmetic materials,
including fragrances. On first approximation, rate of penetration appears
commensurate with compound polarity, measured as the octanol/water
partition coefficient Poct, or its logarithm, log P (vide infra). Molecular
transport of compounds has been described by mathematical models derived
through multiple regression analysis of percutaneous absorption data and
physicochemical constants of a large number of structures. Several mathematical
models descriptive of molecular transport through human skin,
in particular, have been developed more recently (21,28–32). Scheuplein
et al. were among the first to describe an anatomically accurate and physicochemically
reasonable model of the skin, and went on to demonstrate
the correlation between lipophilicity (polarity) and experimental permeability
coefficient values Kp of permeants in vitro (12,18,33,34). Polarity is most
often expressed as the compound’s partition coefficient in equilibrium
between n-octanol and water, Poct. Thanks to these modeling efforts, it is
now possible to predict diffusion of nonelectrolytes through the skin as a
model membrane with some accuracy, simply based on a few physicochemical
parameters that characterize the permeant. Such models are useful in
predicting the activity of chemicals in the absence of experimental data on
a particular compound, without the need for its actual synthesis. Set in
relation to the corresponding in vivo data, the predictive power of these
models, known as quantitative structure–activity relationships (or QSAR),
demonstrates that (i) three descriptors (size, polarity, and hydrogen bonding)
are the principal determinants for cutaneous transport, and (ii) the
properties of the intercellular SC lipids alone are sufficient to characterize
the barrier properties of the skin as the route of permeation for that category
of chemical structures (32).
Rougier et al. developed a brief experimental method to predict skin
absorption of lipophilic compounds. By that method, the systemic uptake
of permeants can be derived from the amount that diffuses into the SC in
vivo after a limited time of exposure (35). The chemical is dosed on the skin
of animals or human volunteers, and after 30-minute-contact the surface of

Basics of Metal Skin Penetration: Scope and Limitations 29
the SC wiped clean of residual compound, and the SC removed by successive
application of adhesive tape and stripping. The strippings are analyzed
for chemical adhering to tissue removed, and from that value the amount
may be estimated that eventually will be systemically absorbed over a longer
period of time.
Certain metal compounds (organometallics) also represent a lipophilic
category (36) that can penetrate the SC with relative ease. Such derivatives
of the more toxic heavy metals, therefore, represent a major toxicological
risk, particularly in the work place, due to their ready skin penetration.
Representative Kps are 2.9 10 3 cm/hr for methyl 203 mercury dicyanamide
50 and 2.3 10 3 cm/hr for lead naphthenate 51 .
Electrolytes
In our present understanding of structure and function of the skin, its permeation
is determined by the physicochemical parameters of permeants.
Intuitively, and also based on the therapeutic action of dermatologicals
and cosmeceuticals, or the toxic effect of skin exposure to pesticides, we
anticipate easy penetration by lipophilic compounds there, as is achieved
in the process of inunction. But for polar structures, such as water or electrolytes
(e.g., salts in aqueous solution), skin penetration to any significant
degree is often dismissed because the skin appears designed as a barrier
against external disturbance of a natural state of hydration. Still, for water
and a number of hydrophilic-charged molecules, including a number of
metal complexes, diffusion is also demonstrated, albeit the process proceeds
at an average of two to three orders of magnitude more slowly than is the
penetration of small-molecular-weight, lipophilic nonelectrolytes, where
Kp values are on the order of 1 cm/hr (e.g., for the solvent toluene).
Skin appendages or shunts (the hair follicles and sweat ducts which
transit through all those layers) are considered the predominant pathway
for the diffusion of large polar molecules or electrolytes across the skin as
an early stage event. They constitute relatively large openings through which
diffusion can occur if two principal conditions are met:
1. Sweat is present to serve as an aqueous diffusion medium
2. the outflow of sweat is significant (18,37)
Measuring electric resistance to analyze for the diffusion pathways
taken by charged molecules, Mali et al. calculated that 70% of the
strong electrolytes permeate through sweat ducts (38). High current flow
in iontophoresis confirmed that observation (12,30,39–43), as did use of
autoradiography or microparticle-induced X-ray emission (PIXE) analysis
(44,45). Considerable experimental data have indicated that the depth of
sweat duct penetration is significantly dependent on ionic mobility (i.e., size)
and electronic charge of the metal (46,47).

30 Hosty´nek and Maibach
Direct or indirect observations speak to the ease of such electrolyte diffusion
through skin shunts. Penetration through sweat ducts can occur
within one to five minutes following exposure, with no comparable transport
occurring via the transcellular path in that time span (48–50). Poral
transit by nickel salts, for instance, can also become manifest clinically;
follicular inflammation or punctate erythema were observed in the context
of skin patch testing with nickel salts (51).
The relatively rapid flux across the appendageal pathway is followed
by slower but continuous, potentially more important intercellular diffusion.
It is reasonable to postulate that metal ion pairs (soaps) formed with
fatty acids on the skin surface will preferably partition into the lipophilic
environment. The intercellular lipid domains in the SC seem to present a
ready pathway for diffusion of such compounds, because the relatively
slower, transcellular rate of penetration does not explain such phenomena
as provocation of allergic reactions due to casual contact with metallic
objects, such as coins.
Occupational health statistics on untoward health effects are a prime
indication for the skin diffusivity of noxious electrolytes. With respect
to specific (allergic) and nonspecific (irritant) contact dermatitis that may
constitute up to 90% of workers’ compensation claims for skin diseases,
percutaneous absorption of potential allergens and irritants is a key determinant
of the risk of skin sensitization and irritation, and may be enhanced
if protective clothing entraps or occludes the irritant against the skin, by
increased hydration of the SC, and by contact with anatomical sites where
skin permeability is greater. Thus, exposure to nickel, chromium, cobalt,
and mercury compounds are premier causes for allergic and irritant reactions
in industrialized countries (52).
The route of transcellular diffusion may be of marginal importance,
especially for electrophilic transition metals that tend to form permanent
deposits on the skin surface. Absorption would be limited to the outermost
layers of the SC, and, possibly, the epithelium of appendages. Such adsorption
may also be terminal, resulting in the depot formation repeatedly
described in the literature for a number of electrophilic metals (53,54). Such
non-Fickian behavior clearly puts that class of compounds beyond the scope
of theoretical algorithms predictive of percutaneous penetration, and the
fate of chemicals thus retained in the SC remains uncertain, prompting
the question: Will the compounds continue to diffuse slowly to be ultimately
absorbed systemically, or will they be shed with the corneocytes in the
process of desquamation? Studies with lipophilic compounds in vivo and in
vitro show that within weeks most of the xenobiotics initially retained in the
SC have been absorbed systemically or have reached the receptor fluid,
respectively. Rougier’s predictive method is applicable only to compounds
of Fickian behavior, but not to electrophilic metals that react with barrier
tissue, rendering in-depth diffusion unpredictable (55). In addition, certain

Basics of Metal Skin Penetration: Scope and Limitations 31
metals, such as copper, zinc, and chromium, are essential nutrients subject
to homeostatic controls, which appear to determine deposition or mobilization
in response to fluctuating body burdens.
Highly protein-reactive, electrophilic metals, such as mercury, to a
large extent were seen to remain at the site of initial absorption. Hursh et al.
measured the uptake of mercury vapor by the skin on human arms in vivo
(56). The rate determined corresponded to a high permeability coefficient
(K p of 1–2 cm/hr). As much as half of the absorbed mercury, however,
appeared to have reacted with SC protein and was eliminated through the
process of desquamation in the following weeks. In that experiment,
the forearm was exposed to mercury vapor enclosed within a saran bag.
On average, 6.8% (range: 3.0–10.6%) of the Hg vapor originally in the exposure
chamber was absorbed by the arm. Up to half the mercury initially
in the forearm was shed by desquamation of epidermal cells during several
weeks. The remainder diffused into the general circulation and could be
measured as systemic mercury. For two subjects on the day following exposure,
the SC was collected from a 35 cm 2 area of the forearm by stripping
off the superficial layers with adhesive cellophane tape. The 203 Hg measured
on the tape, normalized for the total exposed area, corresponded to only
0.3% and 1.3% of the Hg on the arm at that time. When the process was
repeated for one subject 14 days later, and again 23 days after exposure,
the additional recoveries normalized to the entire exposed area were 24%
and 10.7%, respectively, reflecting gradual outward migration of keratinocyte
and corneocyte carriers of the irreversibly bound mercury. The authors
concluded that, due to protein reactivity, absorption of mercury vapor by
the skin poses a minor occupational hazard when compared with inhalation.
The three modes of diffusion available to xenobiotics also illustrate the
characteristics of the skin, which can function to varying degrees as a barrier,
a reservoir, and/or a filter, depending on the polarity and chemical reactivity
of the solute. Topically applied compounds can penetrate the SC along more
than one pathway simultaneously, albeit at different rates. A molecule can
follow any of these routes—follicular, transcorneal, or intercellular—but it
is difficult to assess the relative importance of each, as this will vary with
the physicochemical nature of the molecule.
COMPOUNDS FORMED BY METALS IN CONTACT
WITH THE SKIN
Direct and prolonged contact of metals and alloys with the skin may result
in electrochemical reactions that release metal ions. Indicative of the process
of skin penetration by certain metals is the onset of skin reactions, mostly
immunological: contact with coinage, tools, jewelry, or articles of daily
use. The majority of allergic reactions to metals involve nickel (57–64)
and chromium (65–73).

32 Hosty´nek and Maibach
Leaching or release of metals from alloys in contact with body fluids,
such as sweat or sebum, is difficult to predict with accuracy. Aside from
immediate environmental factors, the microenvironment within the particular
alloy is a principal determinant, due to the action of electromotive forces
generated by the presence of other metals (37). Review of published data on
corrosion shows that release rates, as determined through leaching tests, do
not correlate with metal content in an alloy (74). Associated metals in
immediate proximity form a galvanic element (or pile), whereby an electron
current flows from the more electronegative (e.g., nickel) to the nobler, more
electropositive one (e.g., copper), resulting in oxidation or solution (‘‘corrosion’’)
of the more electronegative metal. Corrosion is an electrochemical
process involving movement of electrons (e ) through the metal from anodic
to cathodic areas and related movement of ions in the electrolyte (sweat). At
metal potentials and pH values that occur in sweat, anodic reactions for
some metal constituents in alloys include
Ni ! Ni 2þ þ 2e ; Cu ! Cu 2þ þ 2e ; Fe ! Fe 2þ þ 2e ; Cr ! Cr 3þ þ 3e
The electrons generated are then consumed at the cathode, most
commonly by reaction with oxygen:
O þ H2O þ 2e ! 2ðOHÞ
Whether and how much nickel is mobilized will depend on the immediate
surrounding material (e.g., copper) present in the alloy. The products of
corrosion may then enter the organism, taken up through the skin, through
the oral mucosa, or by the gastrointestinal (GI) tract (75). Transported by
blood throughout the organism, copper, in particular, will be sequestered
by metallothionein (MT) or coeruloplasmin, and deposited in various
organs and tissues (76).
In order to discuss the phenomenon of skin penetration by metal compounds
as ionized salts, complexes, or organometallic compounds, and to
correctly interpret the meaning of experimental results and clinical observations
attributed to the action of metals, it is important to visualize the
structure and function of the skin as a membrane, which can act as barrier,
filter, reservoir, or an outright port of entry, and the microenvironment prevailing
on its surface. Most direct evidence for formation of skin-diffusible
oxidation products on skin contact is the facile elicitation of allergic reactions
by elements, such as nickel (77), and detection of that metal deep in
the SC upon skin contact (78).
The chemical environment encountered on the skin surface explains
the process of solubilization and, subsequently, diffusion upon contact of
metals in their elemental state. By the action of salts and acids present in
sweat and sebum, metals can be converted to a hydrophilic or lipophilic
derivative, respectively. Only then do they become diffusible via the transcellular,
intercellular, and transappendageal route. Skin exudates are secretions

Basics of Metal Skin Penetration: Scope and Limitations 33
of varying composition, covering the epidermis with a film that complements
the skin’s barrier function. That film consists of two components of
endogenous origin, sweat and sebum, secreted by the respective glands,
which have the ability to corrode (oxidize and dissolve) metal surfaces on
contact. Their composition varies in function of physical, pharmacological
and environmental conditions, gender, age, sweat rate, body site, and
method of collection. An abundance of eccrine sweat glands occurs widely
distributed over all exposed skin areas, to dissipate body heat through evaporation.
Besides these occur sebaceous sweat glands, closely associated with
hair follicles, which produce an oily secretion, the sebum (79). The pathways
that oxidation products follow in the process of diffusion through the skin will
depend on the polarity of the salts formed with exudates, and can be predicted
in light of earlier investigations of skin penetration by xenobiotics. Metal
salts in dissociated ionic form do not penetrate the skin as readily as their
unionized (e.g., organometallic) form. Being unionized, they are more lipid
soluble and that seems to be the decisive factor for intercellular, relatively
facile diffusion. Organic salts may penetrate in the form of ion pairs, and also
exhibit relatively high fluxes. For a molecule that is dissociable in water, the
dissociation constant and pH of the immediate environment will determine
degree of ionization and, thus, permeability. Some organometallic compounds,
such as derivatives of lead (80), mercury (81), or nickel (unpublished
data), have actually been characterized as relatively good skin penetrants.
Copper metal reacting with fatty acids may thus also form lipophilic compounds
of marked diffusivity.
Sweat
Values for the main components of sweat have repeatedly been investigated
over time, yielding increasingly accurate data reflecting improvements
in analytical techniques. The main categories of solutes are discussed here.
Listed in Table 1 are the prevalent ranges of eccrine sweat; they are approximations,
as values are unavoidably subject to variation even in normal
Table 1 Mean Levels of Eccrine Sweat Components
Sodium Men 51.9 mEq/L
Women 36.5 mEq/L
Potassium Men 7.5 mEq/L
Women 10.0 mEq/L
Chloride 29.7 mEq/L
Urea 260–1220 mg/L
Lactic acid 360–3600 mg/L
Amino acids 270–2590 mg/L
Ammonia 60–110 mg/L
Source: From Ref. 82.

34 Hosty´nek and Maibach
subjects, due to the type of stress applied to sweat stimulation, ambient and
body temperature, environmental humidity, diet and nutritional status, age,
gender, sweat rate, skin area of collection, local skin temperature, muscular
activity, etc.
The validity of these values in adults is often questioned, however, due
to differences in methods of analysis and sweat stimulation (physiological,
physical, or pharmacological). The main cause for corrosion of metal surfaces
from skin contact in individuals referred to as ‘‘rusters’’ is not due to
elevated electrolyte concentration, as generally assumed, but rather seems
to coincide with palmar hyperhydrosis in those individuals. ‘‘Rusters’’ exude
palmar sweat at inordinately high rate and a low pH, with the pronounced
corrosive action on metal surfaces observed. When the sodium concentration
measured in normal subjects was compared to that of ‘‘rusters,’’ no
significant difference could be observed (mean values of 49.6 mEq/L vs.
49.1 mEq/L, respectively) (83). However, the pH of sweat was measured
to range between 2.1 and 6.9 (57).
Both proteins and amino acids are normal components of mammalian
sweat (84). Quantitative analysis of amino acids has become routinely possible,
thanks to ion-exchange chromatography; however, the data documented
for men by authors differ significantly, probably due to differences in the
stimulation methods applied, to regional differences in anatomical site, or
to the methods used for sampling. Substantial variations were observed in
relative concentrations of amino acids in sweat collected from various body
parts. Amino acids excreted in sweat are independent of dietary intake (85),
although their concentrations increase markedly in blood and urine on oral
protein intake. No differences in amino acid patterns were seen between
young and middle-aged adults, or between men and women (86).
Sebum
Acid components making up the sebum also play an important role in solubilizing
(‘‘corroding’’) metal surfaces. Human skin features an acid mantle of
pH 4 to 6 at the surface of the SC, which increases with depth to pH 7 at its
juncture with live tissue (87). Determinants of this pH are protons that originate
in the epidermis or as products of sebaceous gland activity, gradually
reaching the surface of the skin. They stem from three classes of compounds:
Amino acids (e.g., urocanic acid and pyrrolidone carboxylic acid)
Alpha-hydroxy acids (e.g., lactic and butyric acid), also present in
sweat
Acidic lipids (e.g., cholesteryl sulfate and free fatty acids, primarily
oleic and linoleic) (17,58,88)
At its point of origin in the live epidermis, sebum as secreted by the
sebaceous glands is a complex mixture of lipids consisting of glycerides,
but no free fatty acids.

Basics of Metal Skin Penetration: Scope and Limitations 35
The occurrence of free acids in the SC and on the skin surface is the
result of hydrolysis of phospholipids and glycerides by lipolytic enzymes
occurring in the sebaceous ducts and on the skin surface, and of bacterial
decomposition. On the skin surface, lipids of epidermal origin contain up
to 20% free fatty acids, with 16% originating in the pilosebaceous glands
(58,88). They consist, to the greater part, of C16 and C18 acids, but their
full range reaches from C5 to C22, with an average length of C16. Such
an acid environment plays a regulating role for SC homeostasis with
relevance to the integrity of the barrier function and regeneration of the
SC barrier (89). It is now accepted that the acid environment on the skin
surface supports a number of SC functions (90), some of which are
control of moisture loss from the epidermis and permeability
barrier homeostasis,
providing resistance to fungal and bacterial infection,
SC integrity and cohesion, and
enzymatic processes regulating corneocyte desquamation.
VARIABLES DETERMINING SKIN DIFFUSION OF
METAL COMPOUNDS
Skin penetration by metals is a multifactorial process, which is not fully
understood, and even less predictable. Contributing to the uncertainty are the
effects of chemical speciation of metallic elements, particularly that of
the transition metals. Also, the skin as target organ presents imponderable
and wide variability. Particularly with ETEs, numerous factors come to bear
(e.g., in vivo, permeability can be subject to homeostasis regulating the overall
organism; in vitro, the sections of skin used for diffusion experiments can
present artifacts). Mathematically derived (mechanistic) models predictive
of percutaneous penetration of organic molecules have been developed
based on in vitro and in vivo data through regression analysis of experimental
results, making the estimation of diffusivity of untested structures
possible. However, similar reduction to a few molecular physicochemical
parameters is not feasible when modeling electrolytes, because movement
through biological membranes is highly element specific. A number of factors
are closely interrelated, and their combined effects are not predictable.
A detailed, in-depth review of parameters determining skin diffusivity of
a large number of metals is given by Potts and Guy (32).
Exogenous Factors
Dose
Rate of diffusion of certain transition metals is not commensurate with
applied concentration. With increasing doses, absorption rates of certain
transition metals can reach a plateau value, then decrease with a further

36 Hosty´nek and Maibach
increase in applied concentration. For others, absorption steadily decreases
with increasing dose. This is likely due to the buildup of a secondary diffusion
barrier as a consequence of electrophilic metals forming stable bonds
with proteins of the skin. Thereby, a depot accumulates in the SC, retarding
further penetration in inverse proportion to metal concentration. In vitro,
this leads to lag times that can be as long as days before any permeant is
collected in the receptor compartment, an observation made mainly with
salts of nickel, chromium, and mercury (54,91,92).
An example of non-Fickian behavior is the diffusivity of nickel salts.
Fluxes of NiCl2 and NiSO4 through full-thickness human skin were compared
by Fullerton et al. (93). After lag times of about 50 hours, in experiments
lasting 144 to 239 hours, occluded NiCl2 entered the receptor fluid about
5 to 40 times more rapidly than (i) NiSO4, (ii) NiCl2 with added Na2SO4,
or (iii) NiSO4 with added NaCl. Without occlusion, the permeation of nickel
was reduced by more than 90%, an indication of permeability increasing
with hydration.
Application of chromate with increasing concentrations does not
consistently lead to higher penetration rates. With chromate doses at concentrations
of between 0.00048 and 0.73 M in guinea pig skin in vivo, absorption
of chromate increased to a maximum Kp of 2.64 10 3 at 0.26 M dose,
reached a plateau, and then gradually decreased below detectable levels (94).
Determined with human abdominal epidermis in vitro, the diffusion
coefficient for chromate ion consistently decreased with increasing donor
concentration (95).
With human full-thickness abdominal skin in vitro, permeability coefficients
for dichromate were also inversely proportional to the concentrations
applied (96).
In guinea pigs, absorption of mercuric chloride applied in vivo reached
a maximum at 16 mg Hg/mL, then decreased to nondetectable levels with
increasing concentrations (59).
Vehicle
The solvent can have a profound effect on permeant solubility and on the
skin membrane, and thus on its barrier properties. Petrolatum, for instance,
is a poor solvent for metal salts, where the permeant remains suspended
in fine particles, affording less-than-ideal uniformity in skin contact; on
the other hand, it has an occlusive effect, which increases skin hydration
and thus promotes diffusion of a hydrophilic compound. Quantitative
absorption of zinc in human skin in vitro showed a distinct dependence
on vehicle. The permeability coefficients comparing fluxes from petrolatum
and those from a hydrogel containing zinc chloride were based upon
72-hour periods. The permeability coefficients for 2.4% zinc formulated in
petrolatum was 0.082 10 4 cm/hr. From the hydrogel containing the chloride
salt, the permeability coefficient was 0.29 10 4 cm/hr (i.e., more than

Basics of Metal Skin Penetration: Scope and Limitations 37
three times higher) (60,61). Zinc oxide was applied on intact human skin
in gum rosin versus a hydrocolloid carrier; after 48 hours penetration of zinc in
human epidermis was more than twice the level as compared to the hydrocolloid
vehicle (62). Dimethyl sulfoxide is a solvent that enhances skin
penetration, as it causes swelling of basal SC cells and disrupts the normal
keratin pattern. Dimethyl formamide and dimethyl acetamide applied in
that solvent were seen to diffuse at a significantly accelerated rate (63).
Of practical importance also is the choice of vehicle in standard
diagnostic skin patch testing for sensitization, with the aim of optimum
release of allergen into the viable epidermis while avoiding allergic or irritant
contact dermatitis, leading to false-positive reactions caused by the vehicle
itself. A method for determining the optimal solvent for diagnostic skin
patch testing of allergens, with the intent of minimizing false-negative reactions,
is their application in different solvents on hypersensitive patients. By
recording the ratio of positive elicitation, the solvent can be identified that
better promotes skin diffusion of the xenobiotic. The reaction threshold
to nickel sulfate was tested in 53 sensitized patients in both water and
petrolatum at equal concentrations (390 ppm) (64). The mean reaction
threshold for nickel sulfate in water was significantly lower (0.43%) than
in petrolatum (0.51%). Also the irritation potential of a chemical can be
assessed by applying it in different solvents in dermatological diagnostics,
aiming to minimize the chemical’s potential for irritation and, thereby,
false-positive reactions. Thus, the irritant reactivity of nickel salts in petrolatum
was greater than in water (97).
Such differences in skin reaction in functions of solvent serve to
demonstrate the effect of the solvent carrier on dermal structures and, thus,
the diffusivity of permeants.
Counterion and Molecular Volume
Diffusion of a cation is necessarily tied to the diffusion of its counterion (i.e.,
diffusion of a metal will also proceed as an ion pair); otherwise, an electrical
potential builds up, which will inhibit further diffusion. Thus, the overall
volume is composed of two factors: the ionic radius of the elemental species
[for example, chromium(III) as cations or chromium(IV) as chromate, the
oxo-complex] and the variable size of the counterion (e.g., chloride versus
sulfate versus nitrate, etc).
Barrier diffusion of charged ions as ion pairs has been characterized as
one criterion in skin permeation (98). Their obvious effect on permeant size,
polarizability, and polarity ultimately find expression in respective diffusion
constants (99,100). As the water content of the SC progressively increases
in the deeper layers, degree of ion (complex) hydration increases further,
to become fully hydrated toward the deeper regions of the membrane.
The resulting increased steric hindrance also leads to a further decrease in
ion mobility.

38 Hosty´nek and Maibach
Experimental data also suggest that diffusivity of metal compounds
through skin appears to correlate with their size and, for a given metal
species, the counterion, thus, represents a determining variable. Own experimental
data from depth analysis of nickel levels in the SC following in vivo
application of a number of its salts confirm such effects of counterion size on
their diffusion (101), an effect further amplified by hydration spheres.
Polarity
The polarity of nickel salts as measured by their solubility in the nonpolar
solvent n-octanol at 22 C increases in highly significant intervals (p < 0.0005)
in the following sequence: nitrate < chloride < acetate < sulfate (101). This
increase in polarity determines the same sequential hierarchy for skin diffusivity
and irritancy (93,97,102).
Nature of Chemical Bond
Going from inorganic, mostly water-soluble ionic mineral salts to lipophilic,
organometallic compounds, bonds increasingly assume covalent character,
and their penetration characteristics approach those of nonelectrolytes. With
lead as an example, the role of ionic bond versus covalent bond is an obvious
codeterminant for diffusion rates, the Kp ranging over four orders of magnitude,
from 10 7 for the acetate to 10 3 for the lipophilic naphthenate (103).
The range of penetration constants determined for a set of lead compounds
illustrates the point of polarity as a determinant for skin penetration. That
ranking of skin absorption in function of polarity was obtained for lead
compounds by Bress and Bidanset, in vitro on human skin at equal concentrations,
expressed as quantity absorbed (Table 2) (103).
The lipophilic category, mainly alkyl and aryl derivatives of the more
toxic metals, thus may represent a major health risk in chemical manufacture
and industrial use, due to their ease of skin penetration. As an
example, minute quantities of the highly toxic organometal dimethylmercury
of previously unknown skin diffusivity were apparently absorbed
Table 2 Skin Absorption of Lead Compounds Through Human Skin In Vitro in
Function of Polarity
Compound (as 10 mg Pb) Amount absorbed (mg)
Percentage of dose
absorbed
Tetrabutyl lead 632 56 6.3
Lead nuolate 130 15 1.3
Lead naphthenate 30 3 0.30
Lead acetate 5.0 0.9 0.05
Lead oxide

Basics of Metal Skin Penetration: Scope and Limitations 39
transdermally that led to the death of a university scientist having handled
the chemical in the laboratory (81,104).
Valence
Ionic radii change with the number of outer electrons (i.e., with their
valence), and thereby also the ion’s electrophilicity and reactivity toward
proteins. These variables also determine the rate of metal diffusion. The
negatively charged chromate and dichromate ions as oxo-complexes of
hexavalent chromium do not bind to organic substances, whereas the electrophilic
chromic ion [Cr(III)] shows a strong affinity for epithelial and
dermal tissues, forming stable complexes that slow the rate of diffusion
(73) or prevent it altogether (96).
Occurrence (induction or elicitation) of allergic reactions in the skin due
to allergenic agents may be considered to be an indicator of skin diffusivity
(i.e., does an allergen penetrate the SC to reach the Langerhans’ cells in
the viable epidermis). In animals, epicutaneous sensitization with trivalent
chromium could be achieved only with difficulty. A 20-fold increase in the
concentration of chromic sulfate [Cr(III)] was required to achieve the same
degree of sensitization as with potassium chromate [Cr(VI)]. On the other
hand, the same degree of contact sensitivity resulted from the intradermal
application of either potassium dichromate or chromic sulfate, demonstrating
that the SC is the barrier inhibiting percutaneous diffusion of trivalent chromium
(105). In a study in which chromium sensitive volunteers were patch
tested with a Cr(III)-containing detergent as well as with dichromate [Cr(VI)],
those identified as chromium sensitive showed a positive response to dichromate,
but none were sensitized to the detergent bar containing trivalent
chromium (106). Differences in diffusivity due to protein reactivity between
dichromate and chromic ions were also noted in vitro with human fullthickness
abdominal skin. The permeability constant for hexavalent
chromium as dichromate was an order of magnitude greater than that of
trivalent chromic salts, overriding the retarding effect of dichromate’s larger
molecular volume (106).
Iron occurs mainly in its trivalent state (ferric ion) in nature, which is
biologically unavailable. As a consequence, several mechanisms have
evolved in the course of evolution to make its absorption by living organisms
possible, and in mammals, iron is primarily absorbed in the duodenum
as the divalent ferrous ion. Nutritionally most important is Fe 2þ bound
in hemoglobin, occurring in organs and muscle tissues, and absorbable
through gastrointestinal membranes. Once it reaches its target organ, iron
is enzymatically oxidized back to Fe 3þ (107).
pH and Solubility
Changes in pH can have an effect on the skin penetration by electrolytes.
From a dermatotoxicological perspective chromic [Cr(III)] salts are the least

40 Hosty´nek and Maibach
problematic because of their poor aqueous solubility, which also decreases
further with rising pH. Diffusivity of chromium as the oxo-complex dichromate
can vary widely, ranging over more than an order of magnitude.
Depending on pH, Cr(VI) exists in either the chromate (pH > 6) or dichromate
form (pH ¼ 2–6). In either form, Cr(VI) is the greater hazard for
human health, due to its heightened membrane diffusivity. The permeability
constants measured for chromate–dichromate were higher in the pH range
8 to 12.7 than in the lower range of pH 1.4 to 5.6. Chromium in the higher
pH range exists as the smaller CrO4 2
ion, whereas in the lower pH range,
the larger, less diffusible dichromate ion Cr2O7 2 predominates (108).
The pH dependence of zinc-oxide absorption was demonstrated by
A˚ gren, where at a pH of 5.4 in vitro, uptake of the compound through
human skin was 21 times greater than at pH 7.4 (8).
Protein Reactivity and Depot Formation
The electrophilic nature of many metals creates pronounced protein reactivity
that can result in depot formation in the SC. Such protein–metal
binding typically occurs with aluminum(III), silver(I), mercury(II), chromium(III),
nickel(II), and the metalloid arsenic(III); it can take place in
all strata of the skin to the extent of building up a secondary barrier and
inhibiting further diffusion (109,110). Such deposits can also be reversible,
however, as is the case for essential elements subject to homeostatic control
(e.g., zinc or copper), which are retained by specific storage proteins,
such as MT or coeruloplasmin, and released again upon physiological
demand (111).
Chromium is an example of the metals that, through nonspecific binding,
causes protein denaturation with formation of permanent depots in the
epidermis; these depots appear to be subject to the countercurrent effect of
continuous sloughing of the outer SC layers, with release of significant
amounts of the metal originally absorbed.
Traces of chromium(III) are tenaciously retained, following exposure
to chromium-containing dermatologicals. Elevated levels of the metal were
seen in the skin up to three weeks, following a single inunction of 0.5%
potassium dichromate in petrolatum (112).
In an in vitro diffusion experiment by Gammelgaard, no chromium
was detected in the recipient phase following the application of Cr(III) as
the chloride or nitrate on full-thickness skin for 168 hours, or following
application of dichromate over 48 hours (105).
The avidity with which aluminum(III) forms complexes with skin and
shunt protein means that there is only superficial penetration into the epidermis.
This is the basis of the antiperspirant activity of certain aluminum
salts, such as aluminum chlorohydrate, which, at the pH of the skin (or
sweat), form insoluble salts and precipitates to effectively form a diffusion
barrier (113).

Basics of Metal Skin Penetration: Scope and Limitations 41
Dermal or systemic long-term exposure to a number of metals through
occupation, therapy, lifestyle, or diet can result in their accumulation in the
body, and their preferential deposition in dermal tissues can become visible
as a characteristic discoloration of the skin. As these metals are not subject
to significant natural processes of elimination, they accumulate over a lifetime
and form permanent deposits in their elemental state, visualized histologically
as metal particles. With the exception of arsenic, however, that phenomenon
does not seem to have any untoward effects other than to alter the natural
coloring of the skin.
Time Postapplication
Deposits formed by electrophilic metals appear as the cause for considerable
lag times of several hours or days as observed in in vitro diffusion experiments.
Before any permeant appears in the receptor compartment, in some
cases, such as with chromium as observed by Gammelgaard in the previous
section, the metal fails to appear at all. According to other observations, diffusion
rates will increase with increases in donor concentration to a point,
level off, and ultimately decrease.
With mercuric chloride in vitro on whole-thickness skin, postapplication
time was the most influential variable for absorption rate, indicating secondary
barrier buildup. The percutaneous absorption of mercury was greatest in
the first period (zero to five hours); it decreased in each successive period to the
lowest rate, usually in the final period (36–48 hours), or sometimes in the nextto-the
last period (24–36 hours). By the end of the experiments, the absorption
rate was 40% of the initial rate, often 25% to 35% and sometimes as little as 1%.
This trend was seen with both guinea pig skin and human skin (114).
Commercial products (emulsions and ointments) containing copper have
been studied over a period of 72 hours (9,61). Whether formulated with copper
sulfate or the organic salt copper 2-pyrrolidone 5 carboxylate, apparent
permeability coefficients over 72 hours were in the range 0.015 10 4 to
0.13 10 4 cm/hr. Noteworthy differences in permeability coefficients were
associated with the period of exposure; the values decreased over successive time
periods from near 1 10 4 cm/hr (zero to two hours) to 0.1 10 4 cm/hr or
less (25–48 hours) and finally to undetectable values in two cases (49–72 hours).
As for percutaneous zinc, absorption rates from commercial emulsions
and ointments, zinc 2-pyrrolidone 5-carboxylate, ZnO and ZnSO4, measured
in vitro with human skin were highest in the first two hours after
application, then decreased to less than a 10th of the initial rates (9,61).
Endogenous Factors
Regional Variation
Skin absorption by chemicals, investigated with steroids and pesticides, is
subject to variation among different sites of the body, decreasing in the

42 Hosty´nek and Maibach
order of scrotum > forehead > postauricular > abdomen > forearm > leg >
back (115–117). Apart from regional SC thickness and shunt density, this
appears to be mainly due to intercellular lipid weight percentage and
composition (118).
Because penetration of electrolytes appears to occur mainly through the
skin’s appendages (12,18,30,42), diffusion in hairy areas may be enhanced,
although absorption was also observed through the palm of the hands devoid
of hair follicles; the route of diffusion there was probably via the sweat
ducts (119).
When investigating site dependence of percutaneous absorption for
a number of organic compounds in vivo, Lotte et al. noted a linear relationship
between penetration and TEWL (i.e., the relationship between
the permeability of the skin to the outward movement of water and inward
movement of permeants) (117).
Following application of nickel chloride on the arm and back of
human volunteers, based on tape strip analysis of the SC, the depth/concentrations
of the two sites were steep though at differing gradients, declining
toward deeper SC layers, with significantly different areas under the curve:
30.8 for skin on the back versus 62.4 on the arm (p < 0.0005), an indication
of heightened diffusivity in arm skin (101).
Age of Skin
The incomplete barrier function observed in infants and young children gradually
increases to values seen in the skin of mature individuals. Neonatal or
infant skin has been found to be more permeable to lipophilic compounds
than is adult skin. Feldmann and Maibach studied percutaneous penetration
of taurocholic acid in vitro through male thigh skin as related to age;
after 24 hours, while skin from a 32-year-old absorbed 8.2%, the skin of a
53-year-old absorbed only 0.3% of the dose (119). One theory associates
such decreased permeability with age as function of diminishing blood
supply (120).
Our own in vitro investigation of permeability to a nickel salt (chloride)
and a nickel soap (di-octanoate), using dermatomed skin under identical
experimental conditions, confirms the decreasing trend in skin diffusivity
with age, specifically between skin from a young (age 16 years) versus an
older source (age 64 years) (unpublished data). While the ratios of diffusivity
values for salt and soap remained the same, advanced age brought a reduction
in rates in excess of two orders of magnitude (121).
Homeostatic Controls
Essential elements such as sodium, potassium, calcium, copper, or zinc present
in the SC, epidermis, and dermis are kept in equilibrium by homeostatic
control mechanisms. These mechanisms play a part in the overall physiological
dynamics of hydration, and also appear to keep the body burden of

Basics of Metal Skin Penetration: Scope and Limitations 43
certain xenobiotics under control. Homeostasis ascertains maintenance of
equilibria necessary for optimal functioning of the organism (e.g., by preventing
undue loss due to perspiration or desquamation through a process of
reabsorption, as has been described for sodium and calcium, in particular)
(122,123).
An important part of such controls are metal-binding MTs, single-chain
polypeptides, present in several organs, including the skin. They have an unusual
amino acid composition consisting of one-third cysteine (20 cysteines in
mammalian MTs) and no histidine, aromatic, or heterocyclic components.
This abundance of thiol groups imparts both reversible metal-binding capacity
and the ability to scavenge free radicals. At least three functions have been
attributed to MTs: metal sequestration, temporary (reversible) binding as well
as long-term storage, and both intra- and extra-cellular metal transport. They
bind the metabolically essential zinc and copper, as well as cytotoxic metals
such as mercury, lead, or nickel.
Levels of free ionic copper, a relatively toxic metal, are moderated
to the minimum levels sufficient for physiologic needs; 10 19 mol/L are
estimated in blood plasma via binding to MT as well as the copper carrier
protein ceruloplasmin (124). An excess of free ionic copper in cells would
lead to oxidative damage, and the affinity of MTs for copper is second only
to that of mercury. The dynamic equilibrium between ceruloplasmin and
MT contribute to the prevention of both toxic accumulation and deficiency
of copper in mammals (125).
When present above specific threshold levels, even ETEs will exhibit
acute toxicity; and by immobilizing excess amounts, MTs fulfill a critical
detoxifying role. MTs thus remove, store, or release metals on environmental
exposure or physiological demand, and are an important factor in homeostasis,
regulating their uptake (diffusion) and release.
Measurements of the rate of skin penetration by zinc have been contradictory,
possibly due to such endogenous controls. Apparently zinc
absorption does not occur by simple diffusion, but seems to be regulated
by MTs (126). MTs thus provide a buffering capacity maintaining intracellular
steady-state kinetics for both copper and zinc, and ensuring a supply of
these metals for other metabolic functions.
As homeostasis is responsible for a rapid exchange between skinapplied
zinc and the large pool of endogenous metal, dermal absorption
experiments conducted in vivo thus should account for natural processes
that may counteract passive diffusion, adding a degree of uncertainty to permeability
constants measured.
Skin Sections
Data are limited on the effect of individual skin layers (SC, epidermis, and
dermis) on percutaneous penetration by metals, and on metal uptake in the
individual strata. An example of the variability observed is the different

44 Hosty´nek and Maibach
penetration rates measured in vitro for nickel chloride. Comparison of Kps
determined through different skin strata by different authors shows that
penetration by nickel chloride is slowest through the SC (37,53,91,93). From
a Kp of 10 7 cm/hr in the SC, the values progressively increase toward fullthickness
skin, with a maximum of 9.8 10 3 cm/hr in dermatomed skin.
Skin sections prepared for in vitro experiments, other than dermatomed
skin, are likely to introduce artifacts to varying degrees. One possible
explanation for low diffusion values through the SC is that shunts in isolated
SC (and to some degree in epidermis) swell shut upon hydration (127). In
vitro diffusion data, which come closest to in vivo conditions, are likely to
be those measured through split-thickness skin. That tissue can be standardized
using the dermatome to a desired thickness (200–400 mm). Unlike other
methods of skin sectioning, use of the dermatome is the best way of preparing
skin for percutaneous absorption studies. A dermatome can be used with
hairless or haired skin, without adversely affecting the viability of the membrane.
Dermatomed tissue includes the layer of dermis where the permeant
is taken up in the capillary system, without adding the variable thickness
of dermal and subdermal tissue and fat.
Skin Metabolism (Red/Ox)
There is evidence of cutaneous metabolic effects on metals of exogenous or
endogenous sources; both oxidation and reduction can occur, manifest in
changes of oxidation state in situ during the process of permeation. The
result can be altered immunogenicity of the metals; examples are known
where oxidation can lead to enhanced immunogenicity of certain metals
[e.g., of Ni(II) to Ni(III) or Ni(IV)] (128), or reduction to lesser immunogenicity
[e.g., Cr(VI) to Cr(III)] (129).
Animal experiments conducted by Artik et al. showed that biooxidation
of Ni(II) to Ni(III) or Ni(IV) occurs through endogenous-reactive
oxygen in the form of hydrogen peroxide or hypochlorite present in inflamed
skin. In animal and cell line tests Artik did find that Ni(II) sensitizes only
na€ve T cells following bio-oxidation to its higher valence, but not the form
of Ni(II) itself (128).
Chromium applied on the skin as chromate or dichromate [Cr(VI)] is
reduced to chromic ion (Cr 3þ ) by tissue proteins containing sulfhydryl
groups (130). In in vitro diffusion experiments with Cr(VI) salts, only chromic
ion is found initially in the receptor phase. On sustained application,
however, Cr(VI) as chromate or dichromate is seen to pass through the skin
unchanged, an indication that a given tissue mass has only a limited capacity
to reduce the chromate ion (105). For some metals, reduction can lead to
discoloration of the skin due to accumulation of the element in the metallic
state (e.g., systemic silver preferentially accumulates in the skin, where it is
reduced to the metallic state). Such impregnation of dermal tissues with
silver deposits results in permanent graying of the skin, a condition termed

Basics of Metal Skin Penetration: Scope and Limitations 45
argyria (131). Chronic dermal application of mercurials used as skin
bleaches (mercurous chloride, ammoniated mercury, and mercurous oxide)
can lead to tissue accumulation of metallic mercury, characterized by slategray
pigmentation or hydrargyrosis cutis (132). The skin is a target organ
for arsenic and is critically sensitive to arsenic toxicity, regardless of the
route of exposure. This is due to the attraction of arsenic to the skin’s sulfhydryl-group
containing proteins. Such accumulation of arsenic in the skin
is characterized by hyperpigmentation, keratoses of the palms of the hands
and soles of the feet, and diffuse macular pigmentation with the characteristic
appearance of ‘‘raindrops’’ or diffuse darkening of the skin on the limbs
and trunk, attributed to the reduction and deposition of the element in the
metallic state (127).
METHODS FOR MEASURING PERCUTANEOUS ABSORPTION
In this context, the terms absorption, diffusion, and penetration are used
interchangeably as they apply to the process of penetrating the outermost
skin layer, the SC, and to all the associated and subsequent events, including
distribution to the different strata and appendages of the skin.
In Vitro Methods
Preferably skin diffusivity of metals is measured in vitro using excised
(animal or human) skin, especially in the investigation of materials of
unknown or obvious toxicity. Reasons for such preference are:
1. Formation of depots in the SC by electrophilic metal ions, establishing
a secondary barrier to further diffusion, which results in
substantial lag times or failure to penetrate the epidermis altogether
(93,105)
2. The need to use radiolabeled materials for the detection of exceedingly
low levels of permeant
3. The pronounced toxicity of some (transition) metals, too hazardous
to apply on the human organism in vivo
Widely used methods for the exploration of percutaneous absorption,
in vitro designs allow a preliminary and early evaluation of safety for
chemicals in the developmental stage, as well as of those too toxic to test
in living models. These methods offer the advantage of yielding data that
reflect the process occurring in selected domains of the skin (SC, epidermis,
and dermis) without involvement of other factors, such as secondary absorption,
deposition, or metabolism in the body’s tissues.
The most commonly used in vitro technique for measuring percutaneous
absorption involves placing a piece of excised skin in a two-chamber
diffusion cell. It consists of a top chamber to receive an adequate volume of

46 Hosty´nek and Maibach
penetrant in solution, an O-ring to secure the skin in place, a temperaturecontrolled
bottom chamber with continually circulating solution removing
the penetrating amounts on the receptor side of the membrane, and a sampling
port to withdraw fractions at specific time intervals for analysis (133).
The solute diffuses from the fixed higher concentration medium in the donor
chamber into the less concentrated solution in the receptor chamber. The
solutions in both chambers are stirred continuously to maintain uniform
concentrations. The advantage of diffusion experiments thus conducted lies
in the applicability of Fick’s first law of diffusion.
In the two-chambered system the two chambers are separated by the
skin membrane. To satisfy the requirements of the Fickian diffusion, useful
for the study of diffusion through biological membranes, an (ideally) infinite
dose is placed in the donor chamber, and appearance of the permeant continually
monitored in the receptor chamber. The solutions in both chambers
are stirred to maintain uniform concentration. Experimental details are
given with an example of steady-state flux measured through the SC (23).
In the one-chambered system, the skin is placed on the chamber and is
open to the environment above, simulating conditions prevailing in real-life
application of drugs and cosmetic products to the skin. As permeation proceeds,
steady-state (Fickian) conditions are not attained (20). The chamber
beneath the skin serves as a container for the receptor fluid that is continually
stirred. Sampling occurs through a side arm for analysis to determine rates of
absorption (134,135). Automatic sample collection is also possible from a
one-chambered cell (136). The receptor volume is small (0.13–0.26 mL) and
allows complete and rapid flushing of the receptor chamber. Special provisions
are necessary to avoid evaporation of volatile compounds from
the surface of the skin. They can be collected through appropriate cell
design (137,138).
Limitations and Problems in Measuring Percutaneous
Absorption of Metal Compounds In Vitro
A limitation in the validity of percutaneous absorption values determined
for electrophilic metals in vitro is the formation of depots in the skin. Such
protein reactivity can result in depot formation in the SC before permeant
diffusion continues into the deeper layers of the skin, and in vitro it can cause
considerable delays (lag times) before the compound emerges in the receptor
phase. For some metals, lag times of several hours or days have been recorded
before any permeant appears in the receptor compartment, or as in some
cases such as chromium, fails to emerge at all. This phenomenon has been
reported for the more extensively investigated metals, such as nickel, cadmium,
and chromium (93,105,139). Due to the electrophilicity of transition
metals, in particular, it can be safely assumed that latency or retardation in
the diffusion process is a predictable phenomenon. As permeability constants
and bioavailability are calculated only from amounts of permeant collected

Basics of Metal Skin Penetration: Scope and Limitations 47
in the receptor phase at steady-state rates, the formation of depots is
rarely part of in vitro diffusion studies, although it should be considered as
part of the total-dose absorbed. The permeant retained in the different levels
of the skin tissues may then eventually become available systemically by
slow diffusion.
For those metals where skin penetration was investigated, the data
rarely are adequate for the calculation of flux or permeability coefficient,
and values can only be derived through a number of assumptions.
Results that are amenable to quantitative analysis often followed
diverse experimental protocols that lead to results that are not necessarily
comparable.
While a permeability coefficient is ideally determined under steadystate
conditions, the percutaneous absorption of metals rarely meets this
criterion.
A problem inherent in the in vitro approach is the lack of total-dose
accountability, as results in the literature are often based on the quantity
of permeant found in the receptor phase only. The amount of chemical collected
in the receptor phase, expressed as percentage of dose, is then taken to
be the amount becoming available systemically, and material retained in the
diffusion membrane (SC, epidermis, and dermatomed or full-thickness skin)
that may subsequently diffuse further, and thus become a significant factor
for risk assessment purposes, is not determined.
In Vivo Methods
The purpose of measuring the percutaneous absorption in vivo is to study
bioavailability of chemicals applied on the skin by analysis of dose moving
into the skin, further into the systemic circulation, and beyond. Such
kinetics measure diffusion by skin tape stripping, bioengineering techniques
such the TEWL, or by traditional analysis of blood and excreta levels.
Taken together, these components can define the dermatopharmacokinetics
of a chemical. After topical application, the levels of xenobiotic in tissues
and body fluids as a rule are below assay detection levels, and it is necessary
to use tracer methods.
The most relevant in vivo data on percutaneous absorption will be
obtained from studies in humans themselves. However, animal models are
needed for the development of basic pharmacokinetic principles and for
the investigation of pharmacological mechanisms. On the basis of currently
available data, the only animals in which permeation is consistent qualitatively
and qualitatively with human permeation data are the weanling pig
(138), the rhesus monkey (136), and the hairless rat (137). Extra body fat
in the pig may affect drug distribution compared to humans, however,
and must be considered. In the rhesus monkey, skin application should be
limited to the nonhairy regions on the ventral surfaces of the animal.

48 Hosty´nek and Maibach
Overall, correlation of animal skin penetration with human absorption data
made by different researchers can vary depending on the compound nature
involved (22). Skin absorption rates determined in animals as a rule result
in a conservative estimate as they overestimate the dose percutaneously
absorbed in humans. Penetration and absorption studies must address toxicologic
aspects and toxicokinetic aspects, as well as developmental and
clinical issues. Also the benefits of using radiolabeled versus nonradiolabeled
analytical methodology have to be considered.
Studies can focus on drug in the skin, in venous blood draining
the application site, in the systemic circulation, or in the excreta. For pharmacokinetics,
drug concentrations are followed over time. For topical
dermatological products, toxicokinetic and safety studies are preferably
performed in the same species.
An indirect method is the quantification of RA in excreta, where percutaneous
absorption is determined by measuring the appearance of RA following
topical application of a labeled compound. Measured is the total RA
of parent compound and any labeled metabolites, which may occur in transit
through skin and body. Label retained in the organism or excreted by
another route (e.g., exhaled air) will not be counted by that method. Therefore,
Feldmann and Maibach used the following expression to correct for
RA unaccounted for:
Percent absorbed ¼ 100 total RA from topical administration/total RA
following parenteral administration
Percutaneous absorption can be measured directly by including material
exhaled and that remaining in tissues at the end of the experiment,
besides excreted material in urine and feces. This sum then gives a direct
measure of absorption (140,141). Because absorption is expressed as the
percent of dose applied, no Kp becomes available using these methods.
In the disappearance method the amount of drug absorbed is assessed
as the difference between the amount applied and that recovered at a given
interval. The method relies on the assumption that the difference between the
quantity applied and that recovered corresponds to the amount absorbed; it
can follow different approaches:
1. Following drug application for a fixed time, the residual formulation
is washed from the skin surface, and the amount removed is
analyzed.
2. The formulation is applied, and the drug content in the outer skin
layers is followed in function of time by spectroscopic or radioisotopic
monitoring techniques.
3. Disappearance (loss) of a radioactive compound from the surface
of the skin and the appearance of RA in the excreta following the
topical application of a labeled compound are followed over time.

Basics of Metal Skin Penetration: Scope and Limitations 49
The total RA in the excreta is a mixture of the parent compound
and any labeled metabolites that may result from metabolism of
the parent in the skin and in the body. The method can involve
single-point measurement, or continuous or periodic monitoring
of compound uptake.
One limitation in this approach is the fact that once the applied
compound has penetrated, its disposition in the organism eludes detection,
especially because the application of radioactive material on men is limited
for ethical reasons. Another problem inherent in this approach, particularly
when involving electrophilic, protein-reactive ions, is that the rate measured
may be a combination of absorption into the systemic circulation and the
dose which is retained in the SC. Frederickson described in detail the problems
arising when measuring absorption of compounds that permeate the
skin slowly (142). As seen in in vitro experiments conducted with reservoirforming
compounds, the portion retained in the surface strata of the SC
is far more important than material reaching the receptor phase, if any
(93,105,143).
Also, depth of penetration through the epidermis remains unknown.
Copper is subject to homeostatic control, and is known to be dynamically
bound to MTs in the skin (93). Thereby, even recovery in excreta may not
afford a relevant mass balance. Advantages of this method are that it
requires small amounts of active formulation (at pharmacologically insignificant
concentrations), it is inexpensive, relatively rapid, and applicable in
clinical studies.
The difficulties inherent in skin recovery, volatility of penetrant, and
errors associated with using the difference between the amount of compound
applied and amount remaining make this an inexact method for the quantitative
determination of absorption rates. The disappearance method has
been used extensively with radiolabeled metal salts, primarily by Wahlberg
et al. To measure the loss of material from the skin surface over time, the
guinea pig is the model mostly used in vitro and in vivo, with a limited number
of experiments conducted on human skin in vitro (144). Consistently
using that technique, Wahlberg and coworkers studied a dozen metal salts
for their skin diffusivity. Although the data on skin disappearance were
obtained over relatively short exposure times, the results are valuable as
benchmarks, internally consistent yielding a scale of relative diffusivities
allowing at least partial validation of other measurements for those metals.
Zinc that is reversibly bound to sulfhydryl storage sites becomes available
for immediate systemic absorption when deficiency develops (145).
Skin conditions due to nutritional zinc deficiency, collectively described
as zinc deficiency dermatoses, have been found to respond promptly to
treatment, and dramatic improvement was seen within days of initiating
transdermal zinc therapy (130,146). Occlusive dermal application of a

50 Hosty´nek and Maibach
concentrated zinc ointment to healthy human volunteers on a normal diet
on the other hand did not lead to a significant increase in serum zinc concentration.
When a similar application was made to the skin of patients
on total parenteral nutrition, a dietary routine that typically results in zinc
deficiency, serum zinc levels were nevertheless maintained at surprisingly
normal and constant values. Based on such observations the percutaneous
absoption of zinc appears to depend on actual zinc status in the overall
organism, and thereby is variable. The close association and interrelationship
between zinc and copper, therefore, would indicate similar variability
in trasdermal diffusion rates measured for the latter, making Fick’s law of
diffusion inapplicable.
These caveats notwithstanding, determined under observance of steady
state and normalized for concentration, by default the Kp is still the most
widely used descriptor of chemical diffusion through the skin. Based on a
number of necessary assumptions, literature data can be transformed so as
to put metal absorption values on a common scale, adequate for purposes
of risk assessment.
Skin Stripping—A Semiquantitative In Vivo Method
Use of adhesive tape to sequentially remove layers of the SC in vivo following
topical application of a compound was proven useful in dermato-pharmacokinetic
and -toxicological research to investigate reservoir formation by drugs
(147). Based on an absorption study of selected drugs in vivo, a relationship
was defined between SC concentration and their eventual total (systemic)
absorption (35). By partial stripping (six iterations) of the SC, the amount
of chemical residing in the SC after 30 minutes exposure time highly correlates
with the total amount of chemical penetration within four days, as determined
by the standard urinary excretion method. It thus becomes possible to make a
predictive assessment of total drug uptake from the analysis of superficial SC
levels only.
Although skin stripping can also be used in vitro, its use in vivo most of
all permits drawing a more authentic profile of the SC, as it avoids problems
otherwise encountered, such as the creation of artifacts and damage potentially
associated with sectioning cadaver skin, storing conditions, or duration
of the experiment (tissue integrity). Rougier’s tape stripping protocol obviates
the need for urinary and fecal excretion analysis, and is applicable to nonradiolabeled
determination of percutaneous absorption, because strippings
remove permeant in quantities adequate for nonlabel assay, such as high pressure
liquid chromatography (HPLC).
Rougier’s correlation, however, cannot be expected to be applicable to
investigation of highly electrophilic, protein-reactive compounds, such as transition
metals, some of which appear to form permanent depots in the SC (e.g.,
nickel, lead, or mercury). For those metal compounds, the direct skin stripping

Basics of Metal Skin Penetration: Scope and Limitations 51
technique followed by inductively coupled plasma–mass spectrometry analysis
was useful in visualizing SC penetration by nickel, in particular (78,101).
The outstanding biological characteristic of nickel, from a public
health perspective, is its immunotoxicity. Nickel started out as a prime occupational
hazard reported from the metalworking and refining industry in the
late 19th century and the first part of the 20th century due to its allergenicity
(139,148,149), but since the Second World War it has become a consumers’
affliction as well, because the metal is now part of most alloys used in the
manufacture of common materials in tools and articles of daily contact.
In a number of dermatotoxicity studies, the incidence of nickel hypersensitivity
noted for women formerly ranged from 10% to 20%, that for men
from 1% to 4%. In more recent studies from a number of countries, a striking
increase in these numbers has been recorded, particularly among women
and children of school age: to 31.9% among schoolgirls in Italy (150), to
43.7% among dermatological clinic patients in Poland (151), and to 30%
among an unselected group of schoolgirls in Finland (152).
To gain insight into the dermatopharmacokinetics of nickel as allergen,
the protocol of sequential adhesive tape stripping was implemented to
examine the penetration characteristics of both the nickel salts and nickel in
its metallic state in human SC and its potential to reach the viable epidermis,
following their application on the skin volunteers. Inductively coupled
plasma-mass spectroscopy (ICP-MS) was used to analyze tape strips (78,101).
For nickel chloride, sulfate, nitrate, and acetate, the depth-penetration
profiles obtained by tape stripping and analysis led to a number of conclusions:
1. Up to 24 hours, most of the nickel dose applied remained on the
SC surface
2. Nickel adsorbed accumulated in the uppermost SC layers, and the
concentration gradient between the superficial and deeper layers
increased commensurate with exposure time and concentration.
3. In a number of experiments, mass balance based on amounts
retrieved from the skin surface and cumulative nickel analysis to
the level of the glistening layer showed a deficit. Within 24 hours
nickel salts thus appeared to penetrate beyond the SC to a minor
degree, possibly via the skin shunts, to become systemically available.
4. While the concentration gradients of nickel adsorbed varied with
counterion, anatomical site, dose, and exposure time, toward the
level of the glistening layer for all variables tested, the depth profiles
converged toward nondetectable levels (

52 Hosty´nek and Maibach
distribution profiles increased proportionally with occlusion time but leveled
off with increasing depth after the 10th strip, to continue at constant levels
to the 20th strip. From application (of a large excess) of metal powder on a
surface of 1.15 cm 2 , the total nickel removed with 20 SC strips after maximum
occlusion of 96 hours was 41.6 mg/cm 2 (101).
The method of SC stripping as applied in nickel research could be used
to investigate skin diffusivity of other metals, such as copper. It would bring
insight into the interaction of that metal brought in contact with the microenvironment
on live skin.
In Vivo–In Vitro Correlation
In validation studies comparing absorption values, good agreement between
in vivo and in vitro methods have been obtained if the studies were conducted
along generally accepted guidelines. The application of a permeant
to the skin in vivo may be more physiologically relevant than the use of
in vitro methods, and in case of disagreement, precedence goes to in vivo
data, although in vivo execution may be subject to error. The results of
in vivo studies are reported as the percent of dose absorbed, whereby appropriate
transformation becomes necessary if a Kp is required.
In vitro absorption values are generally good predictors of the rate or
extent of percutaneous absorption in the intact animal. However, the factors
described may affect the predictive accuracy of in vitro absorption methods,
especially for compounds of high hydrophilicity or lipophilicity. Failure to
account for these variables will lead to poor correlation between in vitro
and in vivo percutaneous absorption. If Kp data are used to estimate percutaneous
absorption, it is necessary to examine the experimental conditions
used if in vitro Kps are to correlate with in vivo data. An example is given
with the skin absorption experiment conducted with boron compounds.
Parallel in vitro and in vivo studies conducted by Wester et al. on the skin
absorption of a number of boron compounds identified substantial and
unexpected discrepancies between the results obtained (153). In the in vitro
study, dosing corresponded to infinite (1 mL/cm) and finite (2 mL/cm)
amounts. These doses correspond to infinite amounts available for absorption
as a theoretical setting to define diffusivity, versus a dose remaining on the
skin to convey realistic in vivo short-term exposure. The latter dose was also
applied on the skin in vivo, which was then allowed to dry. The Kp values
calculated from the infinite dose exposure was 1000-fold higher than the
one from the in vivo study, while the finite in vitro dosing mode was only
10-fold higher. The authors concluded that the in vivo and the in vitro finite
dose data appeared more relevant for a real-life exposure scenario, reflecting
that under infinite dosing over 24 hours in vitro the skin membrane was progressively
becoming more permeable. The infinite in vitro dosing protocol
would only be relevant for a full-body exposure over an extended period.

Basics of Metal Skin Penetration: Scope and Limitations 53
ANALYTICAL METHODS FOR METAL DETECTION
The toxicology of metal compounds is becoming increasingly important in
the fields of environmental, occupational, and clinical medicine. The basis
for studying metal toxicity lies in the application of sensitive and accurate
analytical techniques, and tissue localization of metals in tissue (skin) contributes
significantly to the understanding of toxic mechanisms. Going beyond
conventional chemical analysis, physical methods now afford sensitivities in
metal detection that are lower by several orders of magnitude, often reaching
below the ppb level, and the quantitative tools of atomic spectroscopy have
brought the most significant advances. Adapted to monitoring of toxic elements
in biological substrates, such as tissues or body fluids, such sensitivity
is essential for the assessment of risk to toxic metals without the need to
resort to radionuclides. Thus, monitoring of metal levels in skin and body
fluids by physical methods can provide adequate estimates of dose absorbed,
indicating the need for corrective intervention in cases of potential exposure
hazard, especially in the work environment.
Inductively Coupled Plasma–Atomic Emission Spectroscopy
ICP-atomic emission spectroscopy (ICP-AES) permits detection of metals at
the trace amount level, obviating the use of radioisotopes. For the detection
of copper, the current quantitation limit falls in the 5 to 10 ppb (mg/L) range,
a factor of 5 above the true instrumental detection limit as defined by the U.S.
Environmental Protection Agency (EPA) (22). Quantitative detection of
metals is accomplished by ionization of elements in inductively coupled
argon plasma maintained by the interaction of a radiofrequency field and
ionized argon gas. In a sample aerosol (e.g., a vaporized metal salt solution)
atoms and ions are activated at 6000 C to an unstable energy state, and as
they revert to their ground state again, they emit light of characteristic wavelength
and intensity that can be measured.
Inductively Coupled Plasma–Mass Spectroscopy
ICP-MS is a technique applicable to mg/L (ppb) concentrations of many
elements in aqueous medium upon appropriate sample preparation of biological
materials. Reliability of the method for elemental analysis is based upon
multilaboratory performance compared with that of either furnace atomic
absorption spectroscopy or ICP-AES. Normal instrumental detection limit
for copper, as for a number of other transition metals, falls in the 0.5 ppb range.
The method measures ions produced by radiofrequency inductively
coupled plasma. Compound to be analyzed, present in liquid form, is nebulized
and the resulting aerosol transported by argon gas into the plasma
torch. The ions produced are entrained in the plasma gas and introduced,
by means of a water-cooled interface, into a quadrupole mass spectrometer.

54 Hosty´nek and Maibach
The ions produced in the plasma are sorted according to their mass-tocharge
ratios and quantified with a channel electron multiplier (142).
Stable-Isotope Inductively Coupled Plasma–Mass
Spectroscopy Analysis
Assessment of the relevance of metal absorption data can be difficult and is
rendered ambiguous by the intake of naturally occurring elements in the
normal diet, reflecting in the skin and in the overall organism.
Thanks to recent developments in analytical techniques and the availability
of artificially generated stable metal isotopes, it is now possible to
overcome those earlier difficulties. By dosing stable isotopes in vivo, which
pose no risk to volunteers, it is now possible to differentiate between the
two kinds.
Using ICP-MS for the analysis of the stable isotope 10 B, the percutaneous
absorption of boric acid, borax, and disodium octaborate over a
24-hour exposure period was determined in human volunteers, unambiguously
differentiating between the amount absorbed through the skin and
that absorbed with the diet (153).
The stable-isotope approach was used to investigate lead metabolism
in normal humans by replacing part of the dietary lead with lead-204 tracer.
Kinetic and metabolic balance studies of the volunteers kept on controlled
diets for six months indicated that approximately two-thirds of assimilated
lead was dietary in origin; the remainder was inhaled (143).
By that approach, absorption of copper and other metals in the skin
now becomes measurable, independent from endogenous natural isotopes
present in the organism.
Atomic Absorption Spectrophotometry
Atomic Absorption Spectrophotometry (AAS) with Zeeman background
correction is the reference method accepted by the International Union of
Pure and Applied Chemistry and the International Agency for Research
on Cancer for trace element analysis (144). The method is based on absorption
of radiation energy by free atoms, characteristic for each element. In an
atomizer, thermal energy (air–acetylene flame) converts the analyte to free
atoms. In the ground state they absorb resonance radiation from a light
source that emits characteristic radiation (i.e., the spectrum of the analyte
element) whereby an electron transitions from the ground state to one of
the empty orbitals at a higher energy level. A portion of this light is thus
attenuated by such resonance absorption in the probe. A photomultiplier
detector measures the change in radiation intensity and converts it into an
absorbance signal. There is a linear relationship between absorbance and
the concentration of the analyte. The technique is intrinsically specific,

Basics of Metal Skin Penetration: Scope and Limitations 55
because the atoms of a particular element absorb only radiation of their
own characteristic wavelength (154).
AAS is the most common technique for nickel analysis in biological
fluids. The sample to be analyzed is digested in acid, the nickel then chelated
with ammonium tetramethylene dithiocarbamate, the chelate extracted with
4-methyl-2-pentanone, and the nickel in the extract measured by AAS, or the
initial acid solution analyzed as such by AAS directly (155). Currently,
the detection range for nickel by AAS in body fluids (urine and serum) is
0.4 to 0.05 ppb (156).
Electron Spin Resonance
Electron spin resonance (ESR) applies to transitions between electronic spin
states in a magnetic field. The field of a probe is swept by a magnet,
and resonance indicates presence of unpaired d-electrons in paramagnetic
transition metals, the triplet state of two unpaired electrons in orthogonal
orbitals, such as in elemental oxygen, or in isotopes, such as 13 Cor 17 O.
If an unpaired electron registers proximity of magnetic nuclei with a nuclear
spin 1/2, the single absorption is split into hyperfine structures, identifying
interacting nuclei and their number in the vicinity of the unpaired electron.
ESR is applied in in vitro analysis of biological materials, and, most
recently, in analogy with nuclear magnetic resonance (NMR), instruments
have been developed that allow in vivo analysis of live tissue. ESR detects
the presence of stable organic species, such as nitroxides; organometallic
and inorganic species with unpaired electrons can be detected in liquid as well
as solid materials. Transient, short-lived species, such as organic free radicals,
must be continuously generated in the ESR probe to maintain a sufficient
steady-state concentration for detection. Low temperature (freezing) and
spin trapping techniques are used to visualize transient radicals in the biological
matrices. To stabilize short-lived radicals, these are trapped with alkyl
nitroso compounds leading to long-lived adducts, making them amenable
to ESR analysis.
Identity and oxidation state of paramagnetic transition metals with
unpaired electrons in their d-shell are detectable by ESR (e.g., Mn 4þ and
Mn 2þ ,Cu 2þ ,Ni 1þ and Ni 3þ ,Ti 3þ , and Fe 3þ ). The nuclear spin of the transition
metal and the natural abundance of the magnetic isotopes assist in this
intent by causing different signal splitting and patterns.
Changes in the structure and properties of biological membranes (e.g.,
skin) under the influence of changing physical parameters or under the influence
of xenobiotic penetrants have been studied by ESR. Perturbation and
conformational changes in natural constituents in the skin, due to experimental
penetration enhancers applied, have been described by incorporating
various (ESR-detectable) stearic acids as spin labels in the lipid bilayers of
human skin (157).

56 Hosty´nek and Maibach
Using skin biopsies, the effects of ultraviolet (UV) irradiation on
epidermal tissue have been studied by ESR spectroscopy and ESR imaging,
providing evidence for the generation of hydroxyl and lipid radicals that
potentially result in oxidative injury in the SC and epidermis (158).
Particle-Induced X-Ray Emission
PIXE analysis with a proton microprobe allows the determination of trace
elements in epidermal strata prepared by cryosection (159). In PIXE a proton
beam activates an atomic electron, lifting it into a higher orbital. When
an outer shell electron falls back to fill the vacancy created, the transition is
measured as the emission of an X-ray photon, characteristic of the excited
atom. The method with an elemental sensitivity approaching 0.1 mg/kg
(0.1 ppm) is also useful in bulk analysis of alloys with multielement detection
capability, and in spatial analysis to localize elements present in a sample
(160). Using this analytical method in their research into skin penetration
by nickel, Forslind et al. and Malmqvist et al. have been able to localize
nickel in the superficial strata of human skin, with a spacial resolution of
3 mm by15mm and a detection limit of about 30 ppm. The concentration
of nickel following exposure to a nickel solution was found to be highest
in the outer parts of the epidermis, mainly in the outermost SC (161,162),
an indication of the metal ion’s reactivity.
SUMMARY AND CONCLUSIONS
While there is a considerable volume of medical and toxicological literature
addressing the topical and systemic effects of metal absorption, the data available
on the rate of their skin diffusivity are few, vary considerably, and are
fragmented and hard to reconcile. In reviewing the determinants for the
diffusion of metals, both exogenous and endogenous, the intent here was to
delineate the numerous interrelated factors involved in that process; a closer
examination of the multitude of variables may serve to explain the difficulties
encountered by investigators in this line of research, and underscores the need
for continued endeavors in that field and for the development of reliable methods
by which to determine exposure toward toxicological risk assessment.
The diffusion process of metals, particularly of the transition metals, is
different from that of small-molecular-weight, lipophilic organic compounds,
such as drugs, solvents, perfumes, etc., which follow Fick’s law of
diffusion and whose Kps can be predicted applying structure–activity relationships.
Development of a mathematical algorithm with which to assess
dermal absorption of metals by a unified approach on the other hand has
not been possible, due to the myriad of variables described here.
For a small number of metals, diffusion has been thoroughly investigated,
primarily motivated by morbidity and mortality, as a consequence

Basics of Metal Skin Penetration: Scope and Limitations 57
of skin exposure occurring intentionally for the purpose of personal embellishment,
or accidentally in the work environment. The process of diffusion
examined from the anatomical viewpoint of the skin barrier and the metallurgical
and physicochemical properties of the permeants are nature given
but, as documented in this review, they present only part of the difficulties
to be overcome when investigating metal diffusion. The other set of difficulties
stems from the experimental approach in examining the process, either
in vitro or in vivo.
In determining skin diffusivity of metals in vitro for toxicological risk
assessment or therapeutic benefit, for instance, it would appear important
not to limit the focus solely on permeant reaching the receptor phase. Analysis
should also include permeant retained in the strata of the skin for topical
bioavailability purposes. In the case of some metals, the more electrophilic
ions in particular, that value may be the preponderant quantity, and a potentially
significant factor for consideration in exposure risk analysis, as it may
be mobilized subsequently through homeostasis. Some metals, such as copper,
zinc, or calcium, form a reservoir that can be mobilized on homeostatic
demand; others again penetrate into the strata by passive diffusion and can
albeit slowly, reach the systemic compartment.
On the other hand, metals of slow diffusivity, such as transition metals,
also encounter a countercurrent effect in the strata of the skin. As that barrier
is characterized by continuous desquamation, with a total turnover over two
to three weeks (corresponding to an approximate ‘‘inverse’’ K p of 10 6 ), bioavailability
of the metal may be less than indicated by absorption in skin tissue,
because epidermal turnover can significantly reduce further diffusion
into the systemic circulation of highly electrophilic compounds.
As illustrated here, the process of skin barrier diffusion by metal ions
is multifactorial, and the rate of their absorption, both in vitro and in vivo,
remains imponderable a priori due to a number of potentially promoting
and retarding processes acting simultaneously.
ABBREVIATIONS
EPA Environmental Protection Agency
ESR electron spin resonance
GI gastrointestinal
HPLC high pressure liquid chromatography
MT metallothionein
NMR nuclear magnetic resonance
QSAR quantitative structure–activity relationships
RA rheumatoid arthritis
SC stratum corneum
UV ultraviolet

58 Hosty´nek and Maibach
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4
Percutaneous Absorption of
Copper Compounds
Jurij J. Hosty´nek and Howard I. Maibach
Department of Dermatology, University of California at San Francisco
School of Medicine, San Francisco, California, U.S.A.
INTRODUCTION
Published and unpublished qualitative, semi-quantitative, and quantitative
data on cutaneous absorption of this essential trace element brought in contact
with the skin in its metallic form and its organic and inorganic species
are reviewed. The different approaches that have been used to investigate copper
diffusion and analysis of results are dicussed, as well as problems associated
with their interpretation. Diffusion constants Kp registered range
from 10 7 to 10 3 cm/h. Recognized as a critical endogenous anti-inflammatory
agent in the normal organism, inflammatory conditions induce an
increased demand in the organism for copper; such demand could potentially
be satisfied by dermal exposure. Role and importance of copper’s
anti-inflammatory activity and the possibility of transdermal dosing is
viewed in light of the metal’s skin diffusivity data now available.
The largest part of the literature published on the biology of copper
addresses its importance and function as an essential trace element (ETE) in
living organisms, its biochemistry, and role in nutrition and clinical medicine.
Little has been published on its potential for skin diffusivity. So far, the only
quantitative cutaneous diffusion rates given in the literature refer to experiments
performed with copper compounds on the cat in vitro and in vivo,
and with human skin in vitro as part of dermatological formulations.
67

68 Hosty´nek and Maibach
Transformation of data from those earlier studies, based on certain assumptions,
lead to estimated Kp values of 10 6 –10 5 cm/hr for the copper salts
tested, values which lie at the lower end of skin diffusivity rates measured
for transition metal salts. Interpretation of absorption data so far must rely
on clinical observations.
Quantitative data are difficult to interpret due to the facile and
dynamic changes in speciation potentially occurring in the membrane environment.
Copper can exert its biological role thanks to the lability of its
outer electrons in the 3d and 4s orbital shells, energy levels which overlap,
allowing the element to switch readily from the monovalent to the divalent
oxidation state. The transition thus requires minimal activation energy, and
the shift between Cu(I) and Cu(II) is rendered favorable. Such facile changes
in valence, noted also for chromium and iron, play a decisive role for the
ions’ rate of transfer across cell membranes and epithelia, including the skin.
Copper absorbed from the gastrointestinal tract is bound to albumin as a
Cu(II) ion or as a Cu(II)-amino acid-albumin complex, and circulates in
plasma bound to ceruloplasmin, albumin, or transcuprein. At the cellular
interphase, copper dissociates from the carrier and is reduced to Cu(I) by
plasma membrane reductase; once it has crossed the plasma-membrane
barrier, it continues in the Cu(I) state (1). Such changes in speciations are
presumed to occur in transit through the skin also.
QUALITATIVE DIFFUSION DATA
Earlier data on skin penetration by copper are circumstantial and qualitative
only, derived from clinical observations, pharmacological activity, reports
from dermatological practice, and observations made through use of spectroscopic
methods (Table 1). In those studies, parameters that would allow
calculation of diffusion rates are not available.
Reaction of Metallic Copper in Contact with the Skin
and Absorption of Derivatives Formed There
In prolonged contact with the skin, metallic copper is oxidized, forming copper
salts with amino acids, or soaps with fatty acids naturally present on the
skin surface. This becomes apparent through the discoloration of the skin
and of the copper-releasing objects in the areas of contact. These copper
complexes formed on sustained skin contact appear to penetrate the skin,
and the anti-inflammatory (AI) activity attributed to wearing of copper
metal is attributed to their action, as Cu(II) in these complexes is considered
to be essential for such an effect (11,12).
Corrosion of copper articles, worn as bracelets, in contact with the skin
or on immersion in natural or synthetic sweat was investigated by Walker
and Griffin; weight losses of 0.1% to 0.8% per month were recorded (4).

Percutaneous Absorption of Copper Compounds 69
Table 1 Clinical Signs and Analytical Data Demonstrating Cutaneous
Copper Absorption
References Experiment Species Detection
2 In vivo, copper oleate Human Urine levels
3 In vivo, copper metal Human Serum level
4 In vivo, copper metal Human Metal loss
5 In vitro Human Electron
microscopy
Warner R.R.,
personal
communication,
1993
In vitro, Cu sulfate Human Spectral analysis
6 In vivo, copper
salicylate (Alcusal TM Rat Inflammation
)
arthritis
7 In vivo, copper
salicylate
(Dermcusal TM )
Rat Inflammation
8 In vivo, lipophilic
copper complexes
Rat Inflammation
9 In vivo Cu metal in Serum, scalp hair,
ointment urine; AA, ICP-
MS
10 In vivo Cu metal Inflammation, urine
level
Abbreviations: ICP-MS, inductively coupled plasma-mass spectrometry; AA, atomic absorption.
Absorption of copper as reflected in serum levels has been observed
following the accidental lodging of the finely divided metal in the skin. From
an initial value of 13.0 mmol/L, copper serum levels increased over four days
to 19.4 mmol/L (average control: 17.34 mmol/L), then gradually decreased
again, with an apparent half-life of over 100 days. The shape of the serum
copper plot suggests prolonged, extensive absorption (3).
Absorption of the metal was determined qualitatively on men and women
after a four-week application of an ointment containing 20% elementary
copper. Serum and urine concentrations were determined by atomic activation
spectroscopy and concentrations in hair by inductively coupled plasmamass
spectrometry (ICP-MS). Serum concentration was seen to have increased
significantly, in urine it decreased, and in hair it remained constant (9).
Diffusion of Copper Compounds
Following in vivo application of copper oleate in a lanolin-petroleum base
over 24 hours to human back skin, a significant increase in urinary copper

70 Hosty´nek and Maibach
levels was observed over several days, to a maximum of 0.32 mg/L; determined
for a normal control, that value was 0.11 mg/L (2).
Alcusal TM (copper salicylate ethanolate) in ethanol-glycerin applied
on rat skin in vivo showed AI and anti-arthritic activity (6). Dermcusal TM
(copper salicylate) in dimethyl sulfoxide applied on the skin of rats with
adjuvant-induced polyarthritis was three times as effective as a similar
Alcusal formulation (7). AI activity for a number of lipophilic copper complexes
and phenols in dimethyl sulfoxide was studied by Beveridge et al.;
particularly miflumic acid applied dermally on rats was found to be effective
in reducing experimental arthritis in those animals (8).
Electron microscopy of skin treated topically with copper acetate
revealed that copper initially was localized in the intercellular spaces.
Subsequently, the membranes of viable cells were also penetrated, with accumulation
of the metal in and around the cell nucleus (5).
By electron probe analysis and analytical electron microscopy, the
occurrence of copper was followed across human skin in vitro following
application of copper sulfate using Franz-type diffusion cells. Copper was
observed to enter the outer stratum corneum (SC) cells, then, due to an
apparent barrier occurring approximately halfway within the SC, the level
fell below the detection limit (0.1% by weight), only to reappear again at
the granular interface. There, copper was seen to diffuse by the intercellular
route through the stratum granulosum to reach the stratum spinosum
(Warner R.R., personal communication, 1993). Analysis of coppercontaining
organic compounds applied by those authors on human cadaver
skin by iontophoresis showed high and uniform penetration into the viable
epithelial cells, with approximately half that level present in the dermis.
SEMIQUANTITATIVE DATA
Semiquantitative evidence for the formation of copper complexes and their
diffusion into the superficial skin strata (epidermis) was obtained by occlusion
of finely divided copper powder on the arm of volunteers, followed by stripping
of the exposed areas. Evidence was thus obtained that, over time, copper
derivatives formed with skin exudates were absorbed beyond the SC and
became available to systemic absorption (Hostynek, unpublished data).
Under occlusion with exclusion of air, up to 72-hour exposure, copper
values detected in the strippings were low, reverting to naturally present
levels in those individuals. Under semiocclusion allowing access of air by
covering the skin with ‘‘breathable’’ tape, however, after 72 hours, 0.4–
1.4 mg/cm 2 was seen at the level of the stratum lucidum, and in individual
volunteers cumulative amounts of up to 84 mg/cm 2 copper were present
(area under the curve [AUC], determined by ICP-MS analysis; Table 2).
These observations confirm the occurrence of chemical action of skin exudates
(sweat and sebum) on contacting metals, such as copper, to form in situ

Percutaneous Absorption of Copper Compounds 71
Table 2 Copper (AUC) Removed with Tape Strips After Semiocclusive
Application of Copper Powder on Human Skin
Occlusion time (hr) Mean AUC (ng/cm 2 )
0 (control) 38033
24 45845
48 55960
72 83974
Abbreviation: AUC, area under the curve.
diffusible salts, such as the chloride, pyruvate or lactate, and lipophilic derivatives
(soaps) of likely diffusivity, as demonstrated earlier for nickel (13,14).
QUANTITATIVE DATA
Bis(glycinato) Cu(II) complex was the first copper compound for which quantitative
absorption data became available (15). A radioactive ( 64 Cu) 0.05 M
solution in physiological saline applied to excised cat skin revealed, after a
lag time, a steady-state transport rate described as Kp ¼ 24 10 4 cm/hr.
The percutaneous absorption of copper from copper chloride and copper
sulfate has been investigated in vitro with human skin (16). Formulated
for therapeutic purposes, these salts were administered together with zinc
chloride and zinc sulfate, incorporated in the vehicles petrolatum and two
aqueous gels. For CuSO4 from petrolatum or from Carbopol TM gel and for
CuCl2 from Metolose TM gel, the apparent permeability coefficients were
similar, 0.032–0.045 10 4 cm/hr (Table 3). For CuCl2 from petrolatum,
the permeability coefficient was somewhat higher, 0.023–0.16 10 4 cm/hr.
Skin penetration of commercial products (emulsions and ointments)
containing copper and zinc was investigated in human, dermatomed abdominal
skin (400 mm) in vitro for periods of 72 hours (16). Whether formulated
with copper sulfate or the organic salt copper 2-pyrrolidone 5-carboxylate,
Table 3 Diffusivity of Copper (with Zinc) Salts Through Human Skin In Vitro
Salt Formulation 10 4 K p a (cm/hr) SD
CuSO4 (þZnSO4) Petrolatum 0.032 0.031
CuSO 4 (þZnSO 4) Carbopol 940 TM gel 0.045 0.057
CuCl2 (þZnCl2) Petrolatum 0.16 0.12
CuCl2 (þZnCl2) Metolose 60 SH TM gel 0.023 0.010
a Permeability coefficient Kp calculated from copper in the receptor fluid.
Source: From Ref. 16.

72 Hosty´nek and Maibach
Table 4 Percutaneous Absorption of Copper Compounds from Various
Vehicles Through Human Skin In Vitro
Exposure (hr) 10 4 Kp a
Formulation Compound
Concentration
(mg/cm 3 ) 0–2 24–48 48–72
Experiment No. 1
Emulsion A CuPC 1 0.57 0.02 0.06
Emulsion B CuSO4 1.3 0.44 0.010 0.015
Emulsion C CuSO4 0.5 1.2 0.03 0.08
Experiment No. 2
Emulsion A CuPC 1 1.2 0.06 0.13
Ointment D CuSO4 0.9 0.8 0.05 0.09
Ointment E CuSO 4 0.5 0.9 0.10 0.12
a Permeability coefficient Kp (cm/hr).
Source: From Ref. 17.
apparent permeability coefficients, were in the range 0.015 10 4 –
0.13 10 4 cm/hr (Table 4). Permeability coefficient values decreased over
successive time periods from near 1 10 4 cm/hr (zero to two hours) to
0.1 10 4 cm/hr or less (25–48 hours) and finally to undetectable values in
two cases (49–72 hours). The vehicles were three emulsions (two commercial
products and a custom-made variant of one of the former) and two commercial
ointments. All contained both zinc and copper compounds. Each
formulation was applied at the rate of 16 mg/cm 2 .
1. Emulsion A: water/oil, containing ZnO, ZnPC, and CuPC
2. Emulsion B: same as A except that CuSO4 and ZnSO4 replaced
CuPC and ZnPC
3. Emulsion C: water/oil, containing ZnSO4, ZnO, and CuSO4
4. Ointment D: containing ZnSO4, ZnO, and CuSO4
5. Ointment E: containing ZnO and CuSO4
The absorption rate of copper generally decreased as the experiments
progressed, and for formulations B, C, and E there was little or no absorption
in the final 24 hours.
After 72 hours exposure, there was a significant increase in the average
copper concentrations (140–430% of control values) in the epidermis for
all the formulations. In the dermis, where control concentrations averaged
just 2% to 3% of the epidermal control values, the treatments significantly
increased the copper contents.
Using human abdominal epidermis, the diffusion of copper sulfate
and copper acetate as 1% aqueous donor solutions was compared in vitro
(Hosty´nek, unpublished data). The receptor fluid, high pressure liquid chromatography–pure
water, was collected up to 96 hours after the application

Percutaneous Absorption of Copper Compounds 73
Table 5 Copper a Diffusion Constants Kp Through Human Skin In Vitro
Compound 10 4 K p (cm/hr) Comments References
Sulfate (þZnSO4) 0.032–0.045 Split thickness 16
Sulfate 8.84 Split thickness Unpublished b
Sulfate 0.027 Epidermis Unpublished
Chloride (þZnCl2) 0.023–0.16 Split thickness 16
Acetate 0.019 Split thickness Unpublished
Acetate 0.066 Epidermis Unpublished
Histidinate 1.03 Split thickness Unpublished
Gluconate 2.28 Split thickness Unpublished
Glycinate 24 Full thickness c
15
Glycinate 2.43 Split thickness Unpublished
GHK d
0.76 e
Split thickness Unpublished
GHK 25.1 e
Split thickness Unpublished
GHK 0.002 Epidermis Unpublished
a
Cu(II) compounds.
b
Unpublished data, Hostynek et al.
c
Cat skin.
d
Cu-glycine-histidine-lysine.
e
Kps from separate sets of cells in the same diffusion experiment.
of the donor solutions. The copper concentrations in the receptor fluid were
analyzed using ICP-MS. The Kps measured at steady-state flux were 2.73
0.29 10 6 cm/hr for the sulfate and 6.05 1.19 10 6 cm/hr for the acetate.
Permeability coefficients measured for aqueous copper sulfate and
acetate through human epidermis in vitro are of the order of 10 6 cm/hr.
For copper compounds formulated together with zinc compounds for
therapeutic purposes, applied on dermatomed human skin in vitro in
various vehicles, the apparent penetration coefficients were in the range of
3.2 10 6 –1.6 10 5 cm/hr (Table 5).
DISCUSSION AND CONCLUSIONS
Toxicological considerations and pharmacological evidence for the effects of
exposure to exogenous copper: Aspects of safety of exogenous copper need
to be put in proper perspective to allay concerns on the part of clinicians
not accustomed with the action of copper compounds in the therapy of
rheumatoid arthritis and other degenerative-inflammatory human diseases.
Data available indicate that copper may have noxious effects only following
chronic oral (or parenteral) exposure to high amounts of the metalloelement,
particularly upon chronic oral ingestion with food (e.g., water) that
supplies the organism with more than 5 mg/kg of copper per day (18) or in
the case of prolonged hemodialysis with systems that introduce into the circulation
the metal from copper-containing, semipermeable membranes or

74 Hosty´nek and Maibach
Table 6 Acute Toxicity and Therapeutic Indices of Copper Complexes
Copper complex LD50 (SC) mg/kg CFE PA
Cupric acetate 350 70 –
Cupric anthranilate 750 106 50 750
Cupric aspirinate 760 100 150 760
Abbreviations: TI, therapeutic index; CFE, carrageenan foot edema; PA, polyarthritis.
Source: From Refs. 20 and 21.
copper tubing (19). Thus, a real risk of copper toxicity may exist only if the
administering amounts of the metallo-element certainly are far greater than
those that can be actually used for therapeutic purposes, by transdermal
exposure in particular.
Sorenson investigated the acute toxicity of AI copper complexes
injected subcutaneously in rats (partly listed in Table 6) (20,21). Comparison
with the chelating agent demonstrated that the copper complexes were less
toxic and damaging to the organism than the parent drug.
Cupric sulfate has been used medicinally as emetic, astringent, and
anthelmintic. The intestinal mucosa acts as an efficient barrier; however,
excessive amounts of oral copper salts may lead to death. In the normal
human organism, an efficient homeostatic mechanism controls copper
activity, as most is firmly bound to alpha-ceruloplasmin, and heightened
exposure does not result in disease.
Investigating the cutaneous absorption of copper in the skin, applied
as copper(I) oxide and in its elemental form in an ointment at 20% over a
four-week period, the authors concluded that dermal exposure to copper
at those levels does not present a risk of systemic toxicity, nor does it have
any untoward effects in the skin exposed (9).
LIMITATIONS IN MEASURING COPPER ABSORPTION IN VIVO
Homeostasis: Formation of depots and thus of a secondary barrier, which
inhibits further diffusion can be reversible or irreversible. Examples of the
first kind are essential elements subject to homeostatic control, e.g., copper
or zinc, which are retained by specific storage proteins, such as metallothionein
or, for the former, ceruloplasmin. They are again released upon
physiologic demand (22). Homeostasis may thus compromise the validity
of experimentally determined Kp values for copper. Salts of copper or zinc
present in the SC, epidermis, and dermis are kept in equilibrium by control
mechanisms as part of the overall physiological dynamics of micronutrients,
which regulate their status as free ions. Homeostasis maintains equilibria
TI

Percutaneous Absorption of Copper Compounds 75
necessary for the optimal functioning of the organism in general, and the skin
in particular, as absorption is increased in the copper-deficient organism and
decreases once optimal copper status is established. Dermal absorption
experiments conducted in vivo thus are bound to reflect such controls, which
may counteract passive diffusion and add a degree of uncertainty to permeability
constants measured.
INTERDEPENDENCE OF SYSTEMIC COPPER AND ZINC LEVELS
Levels and activity of zinc in the mammalian organism are closely related to
tissue levels of copper. By inference, it is likely that dermal copper absorption
is also a variable in the function of prevailing zinc levels, and some
observations made as to physiological dynamics of zinc by inference may
reflect on copper also, based on such interrelationship of these two metals.
Measurements of zinc absorption have been contradictory. Apparently its
absorption does not take place by simple diffusion, being regulated by
homeostasis, whereby uptake is inversely related to the individual’s nutritional
status and the actual body burden of the metal. Because copper
and zinc coexist in a competitive relationship, excessive levels of zinc as consequence
of dietary supplementation for therapeutic purposes appear to
determine deficiency in copper, resulting in untoward health effects. The
normal serum copper/zinc ratio of 0.9 to 1.2, for example, decreased to
0.5 in consequence of long-term dietary supplementation with the latter (23).
As an electrophilic metal, copper(II) is highly reactive with electronrich
biological tissues (24) and as applies other transition elements, its
in-depth penetration beyond the superficial layers of the SC will be retarded
by reactivity with sulfhydryl groups in cystein, glutathione, or thioglycolic
acid, which abound in skin tissues.
Limitations in the validity of percutaneous absorption data determined
for copper in vivo are thus due to (i) depot formation, (ii) homeostasis, and
(iii) the interdependence of copper and zinc.
As with most transition metals, skin penetration by copper is subject
to numerous interrelated factors, and its nature as an ETE makes diffusion
experiments difficult and interpretation of results uncertain. This may be an
additional reason why in-depth investigation of copper’s dermatopharmacokinetics
is virtually nonexistent.
Overall evidence on skin penetration by metals available so far points
to a few commonalities, be it in respect to the individual xenobiotic or to
characteristics of the skin barrier and the underlying organism; this makes
a few generalizations and anticipatory statements possible, putting copper
diffusivity in perspective with other transition metals. A challenging determinant
in transition metals is the variability in their speciation, which is
critical for their diffusivity through biological membranes. Mineral salts of
copper (electrolytes) in their ionized form, however, traverse the skin barrier

76 Hosty´nek and Maibach
with permeability coefficients of the order of 10 4 –10 6 cm/hr. For comparison,
the Kp for water has been determined at 10 3 cm/hr.
The present literature review covers only studies conducted by us and
others on normal, healthy, and intact skin, not preconditioned in any way.
The only quantitative data on skin penetration by copper or its compounds
through human skin in vivo or in vitro so far is that acquired in our laboratory
(Table 5).
The information generated in our laboratory and by others earlier on
diffusion characteristics permits the following conclusions.
1. Copper compounds and copper applied as the metal do penetrate
the stratum corneum; however, data on their penetration as measured
so far differ widely and, thus, rate and degree of absorption
remain unpredictable.
2. Transepidermal (primarily intercellular) permeation pathways have
been observed for copper compounds. Penetration via hair follicles,
sebaceous glands, and sweat glands has also been established.
3. Lipophilic organo-copper compounds are more easily absorbed
when compared with coordination complexes and salts.
4. Brought in contact with the skin in the elemental state, copper is
oxidized by exudates present on the skin surface, and the resulting
ion penetrates the SC (as the derivative salt or soap) in a timedependent
fashion.
5. In terms of biological functions, the degree and rate of copper compound
penetration is influenced by: (i) homeostatic mechanisms,
(ii) formation and/or pre-existence of a reservoir, and (iii) regulation
by specific binding proteins (metallothionein or ceruloplasmin).
RECOMMENDATIONS FOR RESEARCH
TO FILL EXISTING DATA GAPS
Evidence is well established for the various functions of copper as an ETE, AI
activity among others. In consideration of the evidence concerning the benefits
resulting from transdermal AI therapy with copper compounds, it would
appear useful to establish a database for the skin penetration of such compounds
using rigorous experimental protocols. That becomes possible by
implementing in vitro diffusion experiments through human skin by accurately
measuring the absorption of copper and its compounds under carefully controlled
conditions of skin membrane selection and preparation, ‘‘permeant’’
concentration, vehicle, area and time of exposure, donor and receptor medium,
and also by analysis of diffusion membrane analysis for retained permeant.
There appears to be a need for research to establish the potential for
dermal absorption of copper through skin-diffusible forms of the metal.
Supplementation in the copper-deficient organism may present much needed

Percutaneous Absorption of Copper Compounds 77
relief for a number of conditions associated with the deficiency in this
ETE: integumentary and skeletal abnormalities, defects in growth and
development, abnormalities in sensory perception (22), the myriad of
manifestations of inflammation and connective tissue disease, such as rheumatoid
arthritis (25). While copper(II) has been administered by traditional
drug delivery systems, intravenously as salts or by intramuscular injection of
Cu(II) complexes, there still appears to be a need for a suitable Cu(II)
complex of ready transdermal transport in order to deliver the element to
intracellular sites where it is needed. Parenteral administration is hazardous
because of local toxicity when long-term therapy is necessary (26). Oral
administration of copper complexes in turn is problematic due to their breakdown
at the low gastric pH, whereby copper ions released from the complex
will be subject to excretion and the highly efficient homeostatic control
mechanism designed to prevent much of the metal to be assimilated (27,28).
While the scarce data reviewed here indicate that copper in the ionized
form may be a permeant too poor to effectively reach the target tissue in
adequate dose, results obtained by Pirot et al. and in our laboratory point
to somewhat higher diffusion rates when skin is exposed to copper complexed
with organic moieties. For a better understanding of the pharmacodynamics
of copper skin penetration, further investigation of its diffusivity
in its different chemical forms would appear useful. In analogy with results
obtained for nickel applied as the metal (14), particularly when associated
with amino acids or skin-identical ligands, topical application of copper as
finely disperse powder for maximum surface activity, e.g., already points to
therapeutically effective penetration rates of lipophilic oxidation products
formed with skin exudates, although oxidation of the metal on the skin to
yield diffusible derivatives may appear to be an ineffective dosage form.
CONCLUSIONS
Observations made in vivo serve to confirm that copper metal on prolonged
contact with skin exudates (sweat and sebum), and in the presence of air forms
in situ diffusible salts such as the chloride, pyruvate or lactate, and lipophilic
derivatives (soaps) of likely diffusivity. The role of the skin as a toxicologically
important route of exposure to environmental agents in general and metal
compounds in particular is hereby underscored. It represents an important
port of entry not only to hazardous materials such as readily oxidized metals
which harbor the potential for serious health effects, but also to essential elements
such as copper, whose release and diffusivity through skin contact has
the potential for beneficial and therapeutic action in the inflamed organism.
These findings may contribute to the acceptance of the long-held belief in
the AI effects of copper metal in direct contact with the skin, in particular, and
also may promote the concept of external AI therapy by patches as alternative
to systemic dosing such as intravenous, intraarticular, or intraperitoneal.

78 Hosty´nek and Maibach
Critical for AI activity may be the supplementation of endogenous copper
with exogenous sources, irrespective of the agent’s nature (i.e., elemental, salt,
complex, or as a covalent derivative), because endogenous formation of the
chelate of maximum biological activity will be formed under homeostatic control.
The mechanism of copper complexes acting as AI agents is not known in
all its details, yet evidence so far points to the possible formation of a unique,
as yet undefined metabolite responsible for the observed clinical AI effect.
GLOSSARY
Absorption Uptake into the organism.
Adsorption Material adhering to the skin surface.
Corrosion Electrochemical process involving redox
reactions in the presence of electrolytes.
Electromotive series Arrangement of metals by decreasing order in
their ability to oxidize.
Ionization potential Energy (electron volts, ev) required to remove
an electron from its atomic orbit, with the
value for the Standard Hydrogen Electrode
set at 0.00 ev as an arbitrarily selected
standard reference.
Ligand Atom, ion or functional group bonded to
a central atom, usually a metal, to form a
complex.
Oxidation Process of electron removal from an atom or
ion (e.g., the increase in the proportion of
oxygen in a compound).
Oxidation potential Electrical driving force toward electron loss,
expressed as a potential value (in electron
volts, ev).
Penetration Passive diffusion process of a solute
through skin.
Permeation Diffusion through one or several layers.
Reduction Process of electron gain by an atom or an ion
(e.g., the increase in the proportion of
hydrogen in a compound).
Speciation Valence state; organic vs. inorganic ligand
bonds.
ABBREVIATIONS
AA atomic absorption
AAS atomic activation spectroscopy
AI anti-inflammatory

Percutaneous Absorption of Copper Compounds 79
AUC area under the curve
ETE essential trace element
HPLC high pressure liquid chromatography
NMR nuclear magnetic resonance
SC stratum corneum
UV ultraviolet
REFERENCES
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2. Schmid R, Winkler J. Über die Kutane Kupferresorption aus einer Kupfer
Enthaltenden Salbe. Klin Wochenschr 1938; 17:559.
3. Bentur Y, et al. An unusual skin exposure to copper; clinical and pharmacokinetic
evaluation. J Toxicol Clin Toxicol 1988; 26:371.
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7:100.
5. Odintsova NA. Permeability of human skin to potassium and copper ions and
their ultrastructural localization. Chem Abstr 1978; 89:360.
6. Walker WR, Beveridge SJ, Whitehouse MW. Antiinflammatory activity of
a dermally applied copper salicylate preparation (Alcusal). Agents Actions
1980; 10:1.
7. Beveridge SJ, Walker WR, Whitehouse MW. Anti-inflammatory activity of
copper salicylates applied to rats percutaneously in dimethyl sulphoxide with
glycerol. J Pharm Pharmacol 1980; 32:425.
8. Beveridge SJ, Whitehouse MW, Walker WR. Lipophilic copper(II) formulations:
some correlations between their composition and anti-inflammatory/anti-arthritic
activity when applied to the skin of rats. Agents Actions 1982; 12:225.
9. Gorter RW, Butorac M, Cobian EP. Examination of the cutaneous absorption
of copper after the use of copper-containing ointments. Am J Ther 2004; 11:
453–458.
10. Fat L, Gyorffy L. Occupational dermatitis due to copper exposure. Proceedings
of the OEESC conference, 2002:51–52.
11. Walker WR, Beveridge SJ, Whitehouse MW. Dermal copper drugs: the copper
bracelet and Cu(II) salicylate complexes. Agents Actions (Suppl) 1981; 8:359.
12. Walker WR, Keats DM. An investigation of the therapeutic value of the
‘‘Copper Bracelet’’—dermal assimilation of copper in arthritic/rheumatoid conditions.
Agents Actions 1976; 6:454.
13. Hostynek JJ. Flux of nickel (II) salts vs. a nickel (II) soap across human skin,
in vitro. Exog Dermatol 2003; 2:216–222.
14. Hostynek JJ, et al. Human stratum corneum penetration by nickel: in vivo study
of depth distribution after occlusive application of the metal as powder. Acta
Derm Venereol (Suppl) 2001; 212:5.
15. Walker WR, et al. Perfusion of intact skin by a saline solution of bis(glycinato)
copper(II). Bioinorg Chem 1977; 7:271.
16. Pirot F, et al. Simultaneous absorption of Cu and Zn through human skin
in vitro. Skin Pharmacol 1996; 9:43.

80 Hosty´nek and Maibach
17. Pirot F, et al. In vitro study of percutaneous absorption, cutaneous bioavailability
and bioequivalence of zinc and copper from five topical formulations. Skin
Pharmacol 1996; 9:259.
18. Aggett PJ, Fairweather-Tait S. Adaptation to high and low copper intakes: its
relevance to estimate safe and adequate daily dietary intakes. Am J Clin Nutr
1998; 6:1061S–1063S.
19. Scheinberg HI, Sternlieb I. Copper toxicity and Wilson’s disease. Trace Elements
in Human Health and Disease, Vol. 1; Zinc and Copper, New York: Academic
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20. Sorenson RJ. Some copper coordination compounds and their antiinflammatory
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5
Diffusion of Copper Through
Human Skin In Vivo
Jurij J. Hosty´nek and Howard I. Maibach
Department of Dermatology, University of California at San Francisco
School of Medicine, San Francisco, California, U.S.A.
Frank Dreher
Neocutis, Inc., San Francisco, California, U.S.A.
INTRODUCTION
Sequential tape stripping was implemented on healthy volunteers to examine
the diffusion of copper through human stratum corneum in vivo following
application of the metal as powder on the volar forearm for periods of up to
72 hours.
Exposure sites were stripped 20 times and the strips analyzed for metal
content by inductively coupled plasma–mass spectroscopy with a detection
limit for copper of 0.5 ppb.
Untreated skin was stripped in the same fashion to determine baseline
copper levels for comparison with exposure values resulting from exposure
in respective volunteers.
This chapter, in part, was reprinted from Hosty´nek JJ, Maibach HI, Dreher F. Human stratum
corneum penetration by copper: in vivo study after occlusive and semiocclusive application of
the metal as powder. Food Chem Toxicol (in press), with permission of Elsevier.
81

82 Hosty´nek et al.
Under occlusion with exclusion of air, up to 72 hours copper values
decreased from the superficial to the deeper layers of the stratum corneum
with gradients increasing commensurately with occlusion time, characteristic
of passive diffusion processes. From the 10th strip on, however, levels reverted
to background values.
Under semiocclusion allowing access of air by covering the skin with
‘‘breathable’’ tape, initial copper values lay significantly above baseline values
and concentration gradients increased proportionally with occlusion time. At
72 hours, from the 10th to the 20th strip reaching the glistening epidermal layer,
copper values continued at constant levels, significantly above baseline values.
The results indicate that, in contact with skin, copper will oxidize and
may penetrate the stratum corneum after forming an ion pair with skin exudates.
The rate of reaction seems to depend on contact time and availability
of oxygen. A marked interindividual difference was observed in baseline
values and amounts of copper absorbed.
Arthritis patients have worn copper jewelry of various types for thousands
of years for the relief of inflammation and musculoskeletal disorders.
Their use continues today as folk remedy. Data documenting statistical significant
amelioration of arthritic conditions in patients wearing copper
objects in intimate contact with skin as compared with the effect of placebo
objects, however, are missing to date.
Indicative of the process of skin penetration by certain metals is the
short-term onset of skin reactions, primarily immunologic or nonimmunologic
irritation, resulting from contact with coinage, tools, jewelry, or other
articles of daily use. Major skin reactions to metals involve nickel and chromium
(1,2). Few reports suggest skin reactions to copper also (3–6).
It remains to be demonstrated that copper metal in contact with the
skin does diffuse through the SC to reach the nucleated epidermis, becoming
locally or systemically available. The critical factor in determining if a metal
will penetrate the SC is believed to be the formation of soluble compounds.
In the presence of oxygen, skin exudates may lead to electrochemical reactions
resulting in metal oxidation (corrosion) to form potentially skin-diffusible
compounds with naturally present skin-identical anions such as chloride ion,
with amino acids or fatty acids present on the skin surface (7,8). Such reaction
usually becomes apparent through the discoloration of the skin of the wearer
and of the copper-releasing objects in the areas of contact.
Besides metallurgical properties of the metal (composition, surface
structure, and finish), occurrence and rate of such corrosion will depend
on individual variables: amount and composition of exudates, pH, temperature,
and the availability of oxygen.
So far, this phenomenon of copper metal dissolution, its uptake into the
SC, and its clinical effects has been supported by a number of investigations.
After immersion of copper turnings in human sweat over 24 hours, the metal
concentration increased by an average of two orders of magnitude from the

Diffusion of Copper Through Human Skin In Vivo 83
natural level: from 1–3.5 10 5 to 0.6–3.4 10 3 M (9). Copper complexes
generated on sustained contact of the metal with animal skin appeared to
exert anti-inflammatory (AI) activity in the animals; that was attributed
to the action of copper compounds formed in situ and their cutaneous absorption
(10). By electron microscopy, Odintsova (11) observed the presence of
copper in intercellular spaces, and ultimately around the cell nucleus in
human skin upon exposure to Cu(II) acetate. Absorption of copper has been
observed indirectly following the accidental lodging of the finely divided
metal in human skin dermatoglyphics. Serum levels were found to increase
over four days, and then gradually decrease with an apparent half-life of over
100 days (12). A strong argument in favor of the therapeutic activity of elemental
copper brought in intimate contact with the organism was provided
earlier in an animal study that demonstrated the AI properties of copper
metal present in the organism: a clear prophylactic effect was achieved when
experimental inflammation failed to be induced in rats in which copper metal
had been implanted two months prior to the experiment (13).
It remains to be demonstrated that copper penetrates the skin upon
exposure to the metal in vivo, and thus potential AI effects could be achieved
by dermal exposure also in humans. In order to arrive at a conclusive assessment
of skin penetration by copper, it appears useful to obtain evidence of
cutaneous penetration on contact by use of quantitative metal analysis.
The purpose of the present investigation into the fate of metallic copper
brought in intimate contact with the skin was (i) to obtain (semiquantitative)
evidence for the formation of copper complexes that diffuse and are detectable
by the presence of copper in the SC, (ii) to trace the path and kinetics of
copper diffusion through the SC, (iii) to assess adsorption and reservoir
formation by the penetrating metal in the SC, and (iv) obtain evidence that
if copper absorbed over time it could penetrate beyond the SC and become
available to systemic absorption. Credence could thus be conferred to the
contended benefits of relief from musculoskeletal disorders attributed to
the wearing of copper jewelry, as the potential for the metal’s activity as an
AI agent by dermal exposure in humans so far is still subject of controversy.
This report illustrates the process of copper diffusion following application
of finely distributed (micronized) copper metal on the skin, through
intimate contact under occlusion and semiocclusion. It thus becomes possible
to estimate actual copper concentration profiles, to the level of the living epidermal
tissue and presumed subsequent uptake by dermal microcirculation.
Determining kinetics and penetration depth of permeants by tracing
the concentration profiles in SC has been rendered facile by using the
virtually noninvasive method of SC stripping with adhesive tape and by
the possibility to detect minute quantities of metals using ICP-MS analysis
(14–19). It thus becomes possible to analyze for the presence of elements
such as copper in skin and other biological materials, with detection limits on
the order of 0.5 ppb (20), making the use of radioisotopes unnecessary.

84 Hosty´nek et al.
Investigated by Schwindt et al. (21), on average each tape strip removes
one layer of corneocytes, of approximate 0.5 mm thickness, and after the first
2–3 strips, for a given skin site and test subject each subsequent tape
strip removes the same amount of SC, down to approximately the 20th strip
(r 2 ¼ 0.99). Although furrows in the SC account for the uncertainty in singlelayer
stripping (22), by progressive SC removal with 20 strips it nevertheless
becomes possible to estimate actual copper concentration profiles to the
level of the living epidermal tissue.
EXPERIMENTAL
Subjects
Healthy Caucasian volunteers between the ages of 34 and 69 without evident
dermatological disease participated after giving informed consent. The
study was approved by the University of California Committee on Human
Research. The volar forearm was selected as study site. Three replicates on
adjacent sites were conducted on the same volunteer.
Copper Application, Occlusion, Decontamination, and Stripping
For the purpose of determining baseline copper values, the naturally occurring
copper in the skin of the volunteers was determined by stripping
untreated skin, with three iterations for each occlusion period, on the volar
forearm of the participating volunteers. For occlusion of the SC, plastic
chambers of 12 mm diameter (1.15 cm 2 ) were placed on the premarked area
of the flexor surface on the arm, the chamber covered with the dressing and
left for the predetermined length of time (24, 48, and 72 hours). The occlusive
and semiocclusive application systems consisted of a plastic chamber
(Hill-Top Res., Inc., Cincinnati, U.S.A.) and micropore semiocclusive tape
(3M Health Care, St. Paul, Minnesota, U.S.A.), respectively.
Copper powder [99.7% 3 mm particle size (Aldrich Chem. Co., Milwaukee,
Wisconsin, U.S.A.)] was applied in triplicate on three volunteers. Prior to
application, skin sites were cleansed with deionized water and dried using cotton
swabs. Copper powder was applied on the volar forearm between wrist
and antecubital fossa. The experiment was conducted during the months
of September through April, whereby the exposed skin of the volunteers
exhibited no noticeable tanning. Twenty-five milligrams of copper powder
were placed on a plastic chamber; the chamber then placed on the premarked
area of the flexor surface of the arm of the volunteer, and left
undisturbed for the predetermined length of time. At the end of the exposure
period, the occlusive materials were removed, the site carefully
washed with a metal-complexing detergent containing a 5% solution of
Extran 300 TM (EM Science, Gibbstown, New Jersey, U.S.A.), using cotton
swabs to remove all traces of metal left on the skin surface, then rinsed

Diffusion of Copper Through Human Skin In Vivo 85
with water. The skin site was dried by tapping with cotton balls and finally
by passing an air stream over the surface for one minute.
Before tape stripping, the area of metal contact was marked with a pen
and a template (Transpore TM tape with a 1.2 cm diameter hole) was applied,
to guarantee that SC was removed only from the application site. After
increasing intervals of occlusion, the area of application was stripped sequentially
20 times after covering the 1.15 cm 2 treated area with a 1 in (6.45 cm 2 )
of the precut adhesive tape strips. Polypropylene tape with a backing of
pressure-sensitive acrylate adhesive (Transpore, 3M Health Care, St. Paul,
Minnesota, U.S.A.) of 2.54 cm width was used for stripping. Constant and
uniform pressure (100 g/cm 2 ) was applied on the tape for five seconds, which
was then gradually removed in one slow draw (17). The tape with adhering
SC were placed individually in scintillation vials, 5 mL of concentrated nitric
acid (70%; EM Science, Gibbstown, New Jersey, U.S.A.) was added and the
vials agitated vigorously for three hours on a rotary shaker (Gyrotory TM
water bath model G76; Edison, New Jersey, U.S.A.). The acid solution
was then diluted 20-fold with water for ICP-MS analysis.
Metal Analysis
Samples were prepared by diluting the samples from the stripping experiment
at 1:10 (volume/volume) using 2% HNO3 immediately prior to analysis.
Standard solutions were prepared by diluting a 1000 g/mL Cu stock solution
with 2% HNO3. A six-point external calibration curve (r 2 > 0.999) was used
for quantitation for both Cu isotopes ( 63 Cu and 65 Cu), with 89 Y being
used as an internal standard. The ICP-MS was an Agilent 7500c spectrometer
operated under He collision gas mode. The instrumental detection
limit was 1000 ng/mL), a Perkin-Elmer 4300 dual view ICP optical emission spectrometer
was used for analysis.
Statistical Analysis
Analyses of the AUC were performed using two-sided, paired Student t-test.
The probability value, P < 0.05, was considered significant.
RESULTS
General
Due to the passive nature of the diffusion process, for all subjects a decreasing
concentration gradient from the horny layer to the subcutaneous tissue
became evident (Figs. 1 and 2). This concentration gradient was steep in
the outermost layers of the SC due to its barrier function (Schaefer
and Redelmeier, 1996) (39) and became less so towards the viable layers
of the epidermis.

86 Hosty´nek et al.
Figure 1 Plots of average values for copper removed by sequential tape stripping on
the ventral forearm of three human volunteers after increasing periods of occlusion,
as assessed by ICP-MS. Data points correspond to strip nos. 2, 3, 5, 10, 15, and 20.
Figures 1–2 show copper content for strips nos. 2, 3, 5, 10, 15, and 20
as a function of type and duration of occlusion, as resulting from ICP-MS
analysis. Individual graphs are presented to illustrate the individually differing
levels of copper naturally present in the skin of volunteers.

Diffusion of Copper Through Human Skin In Vivo 87
Figure 2 Plots of average values for copper removed by sequential tape stripping
on the ventral forearm of three human volunteers after increasing periods of
semiocclusion, as assessed by ICP-MS. Data points correspond to strip nos. 2, 3,
5, 10, 15, and 20.
Copper Penetration Under Occlusion
For purposes of range finding, occlusion was initially set at periods from
minutes to 24 hours, as in earlier studies on nickel (23). However, over that

88 Hosty´nek et al.
period copper values did not exceed background values, possibly due to
greater corrosion resistance of copper on skin contact.
Figures 1A–C show copper values in sequential SC strips in terms of
amount per unit area of tissue, presented for individual volunteers and
increasing periods of occlusion. Notable in individual tracings are the
differing baseline levels of copper naturally present in the skin of volunteers.
Only the initial three strips were analyzed for baseline.
For all periods of occlusion, initially profiles were slightly elevated
above baseline. By the 10th strip, however, copper levels had reverted to
baseline values.
Copper Penetration Under Semiocclusion
Figure 2 shows copper values in sequential SC strips in terms of amount
per unit area of tissue, recorded for individual volunteers and increasing periods
of occlusion. Again, the individual tracings show the differing baseline
levels of copper (strips 2–20) naturally present in the skin of volunteers.
Copper values decreased from the superficial SC layers to the deeper layers,
the gradients increasing commensurately with occlusion time (24, 48, and 72
hours). After the 10th strip, the copper content in the SC approximated
baseline levels, but at 72 hours occlusion continued above baseline values.
The AUC values for the semioccluded experiments and their statistical
significance are given in Table 1.
DISCUSSION
Results of this Study
In our study of copper metal exposure, data obtained for the participating
volunteers reveal a striking interindividual diversity in copper levels naturally
occurring in the same anatomical area of the SC, determined before
application of copper powder. Also the variable rate of SC penetration by
Table 1 Copper Removed from Human Volar Lower Arm After
Semiocclusion
Occlusion time Mean AUC a (ng/cm 2 ) SD p-value
0 38,033 18,086
24 45,845 0.11
48 55,960 0.03 b
72 83,974 0.08
a Average area under the curve (AUC) of triplicate stripping experiments on
three volunteers removed by strips nos. 2–20.
b Significant difference in copper removed after 48 hours versus background.

Diffusion of Copper Through Human Skin In Vivo 89
the metal appears due to individual differences in metal oxidation in the
microenvironment prevailing in the area of contact (chemical, i.e., exudates
composition and sweating rate, and physical, i.e., ambient and skin
temperature). A literature search for copper levels in the SC provided no
information that could be used for direct comparison with our results. Only
data on whole skin and hair were found.
The normal copper concentration in healthy human skin tissue appears
to vary according to anatomical site from 1 to 7 ppm dry weight, as determined
by neutron activation analysis of biopsies from 15 individuals (24).
The levels and distribution of copper in human skin (dermis and epidermis)
was also investigated (25). The mean values determined by atomic absorption
in dry tissue varied from 0.88 to 2.60 ppm, with a median value of 1.38 ppm.
Using nuclear microscopy, hair from three normal subjects showed copper
concentrations of 18 6 ppm in the cortex of hair, with marked increases
in concentration at the periphery (approximately 100 ppm) (26). AA spectroscopy
was used to analyze for copper content of hair in relation to age.
Starting at 13 ppm at age 2, copper content increased to 60 ppm by the
age of 12, and decreased again to levels of 10 ppm at age 80 (27).
Discussion of natural levels of that essential trace element prevailing in
the integument and, in particular, of those encountered in the volunteers
participating in the present study lies outside the scope of this paper. Interindividual
variability in the SC diffusion by copper, essentially going back to the
rate of oxidation in the microenvironment of an individual’s skin, on the other
hand, is readily explained, based on previous observations (28,29). Oxidation
of a given metal in contact with the skin is a multifactorial process, as it will
vary in function of prevailing environmental conditions, the skin’s pH, sweating
rate, and the variability in the individual’s amount and composition of
sebum, sweat, and salts, which is, in turn, a function of gender and age (30–36).
The soluble metal complexes formed are capable of penetrating the
corneocyte envelope, forming a reservoir of the metal in the outermost
layers. The process of oxidation appears to be slower under occlusion,
due to limited access of air (oxygen). As became evident from these in vivo
experiments, availability of oxygen is one critical factor in the oxidation process
of metals. In the presence of sweat as electrolyte, the electrochemical
oxidation of copper leads to the formation of cupric ions (Cu 2þ ). This is
coupled with the concurrent reduction of oxygen, which with water forms
hydroxyl ions. In the absence of oxygen, the reaction cannot proceed and
cupric ions are not liberated. One observation in this study points to the difference
in ionization potential and thereby corrosivity of copper when compared
to that of nickel described earlier (29).
In general terms, oxidation of a metal to the ionic form and liberation
of electrons proceeds according to Eq. (1):
M ! M xþ þ x e
ð1Þ

90 Hosty´nek et al.
and the reduction of the (obligatory) oxygen present to form water is represented
in Eq. (2):
O2 þ 4H þ þ 4e ! 2H2O ð2Þ
The difference in corrosion rates between the two metals is expressed
as the standard oxidation potential in the equations below, characterizing
the oxidation for elementary nickel [Eq. (3)] and elementary copper [Eq. (4)],
respectively. While nickel, brought in intimate contact with the skin under
occlusion, reacted to form skin-diffusible derivatives within minutes of
exposure, copper seems not to significantly diffuse for up to 24 hours. This
may be explained by the differences in oxidation potential between nickel
(þ0.23 ev) and copper ( 0.34 ev), nickel thus being more easily oxidizable.
This should not come as a surprise since copper belongs to the select group
of the so-called noble, i.e., nonreactive metals.
Nið0Þ !Ni þ2 þ 2e þ 0:23 ev ð3Þ
Cuð0Þ !Cu þ2 þ 2e 0:34 ev ð4Þ
Other metals besides copper, particularly when they are in intimate
contact with other metals in alloys forming a galvanic element, are electrolytically
oxidized by the skin’s sweat and sebum, forming skin-diffusible
compounds: e.g., nickel and references therein; beryllium, cobalt, chromium,
and mercury, and references therein (28,29). At the other end of the spectrum
in the electromotive series, under specific circumstances skin reactions
have also been attributed to gold in contact with skin, a metal even less reactive
than copper (37). Positive patch test reactions were recorded in patients
with facial and eyelid dermatitis that were ascribed to gold released from
jewelry, apparently promoted by sweat and associated with the abrasive
action of titanium dioxide in cosmetics and sunscreens. A contributing
factor is presumed to be static absorption of loosened metal particles to titanium
dioxide, which can act as adjuvant promoting penetration of skin
strata (38). Nederost and Wagman concluded that a subset of gold-allergic
patients would benefit from avoidance of gold jewelry coming in contact
with skin to which cosmetics were applied containing titanium dioxide and
becoming subject to substantial friction.
The directionally increasing slope of copper concentration profiles with
increasing time of exposure, traced up to 72 hours of occlusion, indicates
a slow but gradual formation of a depot in the outer SC due to its barrier
function (39). Such concentration gradients are typical for a passive diffusion
process, as there are no active transport mechanisms involved through
skin. Copper ions are highly electrophilic and readily complex with SC
proteins, leading to such depot formation. Such depots in the SC merit
consideration for purposes of risk assessment. In vitro diffusion experiments
with several copper complexes showed that a substantial portion of the

Diffusion of Copper Through Human Skin In Vivo 91
permeant is retained in the SC, epidermis, and dermatomed skin used for
that purpose (Hostynek JJ, unpublished data). Such buildup in the SC is
a fair indication that the exogenous agent (copper) eventually may become
available systemically. For that reason also, measuring diffusion rates only
presents part of the overall picture, as the chemical absorbed into the SC
may continue to diffuse into the viable tissues, even after exposure has
stopped. Further absorption of such material, appropriately referred to as
the SC reservoir (40), is the result of a counter-current process that includes
desquamation versus release.
Results confirm that absorption of copper into the SC does occur.
Because copper levels above baseline values, albeit approaching background
and after prolonged semiocclusion, are still seen at the level of the 20th strip
(0.4–1.4 mg/cm 2 ), the level of the stratum lucidum. This is taken as an indication
that the metal has penetrated beyond the deepest SC layers, and thus
is likely to have reached the viable epidermis.
Although extremely fine particles may penetrate the loosely packed
superficial stratum disjunctum, they would not be expected to migrate
further into the deeper, tightly packed cells of the stratum compactum.
Literature on human skin penetration by particulates indicates that follicles
will be penetrated up to a size of 50 mm, and that the SC by an optimal diameter
of 5 mm, migrating to a depth of ca. 10 tape strips, as observed by
optical methods (41–44). In the studies cited above, the intent was to promote
depth penetration of a dermatological preparation applied, achieved
by massage of the treated skin area. Particles lodged in hair follicles would
not appear as artifacts since tape stripping of skin only removes SC without
associated hair follicles (22).
Toxicological Considerations and Pharmacological Evidence
for the Effects of Exogenous Copper
Toxicity of the ionized form of this essential trace element is kept in check
by an efficient homeostatic mechanism involving metallothionein and ceruloplasmin
(45). Data available indicate that copper may have noxious effects
only following chronic oral (or parenteral) exposure to high amounts of the
metallo-element, particularly upon chronic oral ingestion with food (e.g.,
water) that exposes the human organism to more than 5 mg/kg of copper
per day (46).
Sorenson (47) investigated the acute toxicity of AI copper complexes,
partly listed in Table 2. Comparison with the respective chelating agent
demonstrated that the copper complexes were less toxic and damaging to
the organism than the parent drug.
In a phase I trial to investigate the cutaneous absorption of copper in
the skin, the metal was applied as copper (I) oxide and in its elemental form
as an ointment (paraffin and Vaseline) under gauze. Based on daily application

92 Hosty´nek et al.
Table 2 Acute Toxicity and Therapeutic Indices of Copper Complexes
Copper complex LD50 (sc) (mg/kg)
on the skin of volunteers over a four-week period, the authors concluded that
dermal exposure to copper in concentrations of up to 20% does not present a
toxic risk (48).
The risk of copper toxicity may exist only by administering amounts of
the metallo-element far greater than those that can be actually used for therapeutic
purposes, by transdermal exposure in particular.
CONCLUSIONS
The present observations serve to confirm action of skin exudates (sweat
and sebum) in reacting with copper metal on prolonged contact and in the
presence of air, to form in situ diffusible salts, such as the chloride, pyruvate
or lactate, and lipophilic derivatives (soaps) of likely diffusivity (8). The
role of the skin as a toxicologically important route of exposure to environmental
agents in general and metal compounds in particular is hereby
underscored. Skin is an important port of entry not only to hazardous materials
such as readily oxidized metals that harbor the potential for serious
health effects, but also to essential elements such as copper, whose release
and diffusivity through skin contact has the potential for beneficial and
therapeutic action in the inflamed organism.
These findings may contribute to the acceptance of the long-held
belief in the AI effects of copper metal in direct contact with the skin in
particular, but also may promote the concept of external AI therapy as
alternative to systemic dosing, such as intravenous, intra-articular or intraperitoneal.
Critical for AI activity may be the supplementation of endogenous
copper with exogenous sources, irrespective of the agent’s nature: elemental,
salt, complex, or as a covalent derivative, as endogenous formation of
the chelate of maximum biological activity will occur by homeostasis. The
mechanism of copper complexes acting as AI agents is not known in all its
details, yet evidence so far points to the formation of a unique, as yet undefined
metabolite that might be responsible for the suggested clinical AI effect.
TI
CFE PA
Cupric acetate 350 70
Cupric anthranilate 750 106 50 750
Cupric aspirinate 760 100 150 760
Abbreviations: TI, therapeutic index; CFE, carrageenan foot edema; PA, polyarthritis;
sc, subcutaneous.
Source: From Ref. 47.

Diffusion of Copper Through Human Skin In Vivo 93
GLOSSARY
Absorption Uptake into the organism.
Coinage metals Copper, silver, and gold, with negative oxidation
potential relative to the hydrogen (standard)
oxidation potential defined as 0.00 V. Their
characteristic electronic arrangements permit
facile oxidation. Possible oxidation states are I, II,
and III. They are often stabilized through
formation of characteristically covalent
complexes.
Corrosion Electrochemical process involving redox reactions
in the presence of electrolytes.
Electromotive series Arrangement of metals by decreasing order in
their ability to oxidize.
Ionization potential Energy (electron volts, ev) required to remove an
electron from its atomic orbit, with the value for
the standard hydrogen electrode set at 0.00 ev as
an arbitrarily selected standard reference.
Metallic elements Characterized by the ability to form cations
(positive ions), by luster, malleability,
conductivity (thermal and electrical) and the
formation of cations (positive ions).
Noble metals Descriptive term used to characterize
electrochemically inert metals, mostly copper,
silver, gold, palladium, and platinum.
Oxidation Process of electron removal from an atom or ion
(e.g., the increase in the proportion of oxygen in a
compound).
Oxidation potential Electrical driving force toward electron loss,
expressed as a potential value (in electron
volts, ev).
Penetration Passive diffusion process of a solute through skin.
Permeation Diffusion through one or several layers.
Reduction Process of electron gain by an atom or an ion
(e.g., the increase in the proportion of hydrogen in
a compound).
ABBREVIATIONS
AI anti-inflammatory
AUC area under the curve
ev electron volt
SC stratum corneum

94 Hosty´nek et al.
sc subcutaneous
ICP-MS inductively coupled plasma–mass spectroscopy
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11:453–458.

6
Irritation Potential of
Copper Compounds
Jurij J. Hosty´nek and Howard I. Maibach
Department of Dermatology, University of California at San Francisco
School of Medicine, San Francisco, California, U.S.A.
INTRODUCTION
Following clarification of frequently encountered terms in dermatotoxicology
we present a synopsis of irritant reactions of the skin to copper and its
compounds: types of untoward skin reactions in general, aspects of human
exposure to copper, and a description of predictive and diagnostic methods
to assess irritancy through bioengineering methods, in vivo and in vitro, in
humans and animals. The review discusses case studies, followed by critical
examination of literature reports, with consideration given to a number of
confounding factors in diagnosis. To a limited extent, the review also discusses
immunotoxicity, copper pharmacology, and therapeutic benefits of exposure.
EXPOSURE TO COPPER
Natural sources of human copper exposure due to volcanic exhalations,
weathering of mineral deposits, and runoff are a minor factor. The major
release of copper stems from anthropogenic emissions, stemming from
This chapter, in part, was reprinted from Hosty´nek JJ, Maibach HI. Review: skin irritation
potential of copper compounds. Tox Mech Meth 2004; 14:205–213, with permission of Informa
Healthcare.
97

98 Hosty´nek and Maibach
major industrial activities such as mining, smelting and refining, agricultural
and industrial use of copper pesticides and preservatives, the burning of coal,
waste incineration, and widespread consumer applications of copper (e.g.,
brake-pad releases). Thus, occupational exposure is preponderant and
mainly through inhalation. Concentrations of copper in the occupational
setting are rarely reported, as the focus there lies mainly on other elements of
greater toxicity. It is thus difficult to relate health effects from those environments
specifically to copper. Most countries limit copper-containing dust to a
range of 0.5–1.0 mg Cu/m 3 , and copper in fumes to 0.1 and 0.2 mg Cu/m 3 (1).
For purposes of occupational hazards in the United States, a limited
number of compounds are recognized as hazardous on cutaneous exposure,
and are identified as such by a ‘‘skin’’ notation in the listing of hazardous chemicals
by the American Conference of Governmental and Industrial Hygienists
(ACGIH) in their listing of threshold limit values (TLVs). The purpose of such
labeling is to raise attention to the fact that cutaneous absorption can present a
significant risk of systemic toxicity. The criterion most frequently used for a
‘‘skin’’ listing is acute animal toxicity from skin absorption, i.e., a dermal
LD50 below 1000 mg/kg. This may be an indication of either rapid skin penetration
or extreme toxicity, or both. The TLV values applicable to copper as
fume are 0.2 and 1 mg/m 3 as respirable dust or mist, for purposes of irritation,
gastrointestinal exposure, or metal fume fever (inhalation). In the 2001 edition
of TLV guidelines, copper does not rate a skin notation (2).
Exposure of the general population to this essential trace element is of
minor importance, limited to normal dietary intake of copper naturally occurring
in plants and meat, and the metal released into drinking water conveyed
through copper tubing. Systemic exposure to copper occurs through its slow
release from dental materials and intrauterine devices (IUDs). Topical
exposure comes from the release of copper in alloys used in jewelry, as it is
measurably released in contact with skin exudates.
SOLUBILIZATION OF COPPER METAL
Dermal
Inflammatory skin reactions of different types are due mostly to exogenous
factors, primarily chemical agents impacting the skin. To exert an irritant or
inflammatory action they must penetrate the stratum corneum (SC), a layer
of inert keratinized cells, before reaching the viable layers of the epidermis
and dermis. Among the irritant chemicals are acids, bases, organic solvents,
salts, soaps and detergents, and pharmacological agents.
Copper and other elements in their metallic state have no effect on the
skin. They become potential irritants or allergens only when they are corroded
(oxidized) and thus become soluble through the action of exudates
encountered on the skin surface, or in a relatively corrosive physiological
environment such as the oral cavity or the uterus.

Irritation Potential of Copper Compounds 99
By the action of salts and acids present in sweat and sebum on the skin,
most base metals are converted to the hydrophilic (ionized salts) or lipophilic
(soap) form, respectively. Sweat composition fluctuates considerably in
function of the rate of sweat secretion (3). Besides sodium and chloride, other
significant corrosive components of sweat are potassium, urea, lactate and
pyruvate, amino acids, proteins, and acidic lipids. The formation of free acids
in the SC and on the skin surface is the result of hydrolysis of those acidic lipids
by lipolytic enzymes occurring in the sebaceous ducts and on the skin surface,
and of bacterial decomposition (4,5). It is the oxidizing (corroding) action of
such acids that results in the formation of soaps with copper (and metals in
general) upon intimate and prolonged contact with articles of daily use, which
potentially result in skin irritation or allergic reactions once they reach the
viable structures of the skin, since these relatively lipophilic compounds penetrate
the SC with relative ease as compared to ionized salts (electrolytes) (6).
Metallic objects used in jewelry or drug-like devices (dental materials,
orthopedic implants) as a rule are not made of copper alone, but the metal
is incorporated in alloys that have corrosion (oxidation) characteristics quite
different from those of the constituent metals. An exception is the wire used in
IUDs, presumably made of high-grade copper only. The characteristics of
alloys are determined by electrochemical characteristics of the elements in
contact with each other; oxidation and formation of potentially allergenic ions
will vary in function of alloy composition. The electrochemical potential (galvanic
effect) between diverse elements in close proximity provide the driving
force for such reactions resulting in enhanced corrosion (7). The more electropositive
(baser) the element (e.g., nickel), the more stable it is in the ionized
state, and will transfer electrons to the more electronegative, nobler metal
(e.g., copper). The actual concentration of a metal in the alloy is thereby only
of secondary importance. Ultimate biological activity of the alloy is determined
by the rate at which metal ions are released, i.e., whether they reach
a concentration sufficient to provoke a reaction in the adjacent tissues.
Release of copper in synthetic sweat related to chloride ion concentration
was determined by Boman et al. After 24 hours, copper dissolved from
coins and copper thread in the range of 80–100 mg/mL sweat, with an inverse
relationship between the concentration of copper and chloride ion (8).
Lidén et al. determined the release of copper from gold-containing
jewelry in artificial sweat. Amounts released over one week ranged between
0.11 and 0.66 mg/cm 2 , depending on alloy composition (9).
Systemic
Corrosion and solution of copper in the physiological environment may be
considered as equivalent to systemic dosing. Human plasma is an aggressive
physiological medium for dissolving metals. Corrosion of the foreign object
in this microenvironment releases components into the organism, some of
which can then act as irritants or allergens. Levels of free ionic copper,

100 Hosty´nek and Maibach
however, a relatively toxic metal, are moderated to the minimum levels
required for physiological needs, 10 19 mol/L estimated in blood plasma,
through binding to ceruloplasmin and metallothionein. The dynamic
equilibrium between ceruloplasmin and metallothionein prevents toxic
accumulation or deficiency of copper in mammals.
Dental Alloys
Amounts of copper released from commonly used dental casting alloys,
measured in cell culture over 10 months, was 0.15 mg/cm 2 /day (10). Cytotoxicity
of metals thus released from dental alloys could be considered a correlate
of irritancy. Accordingly, Wataha et al. (11–14) investigated in vitro corrosion
rates of dental casting alloys in various culture media to obtain a measure
of biological risk to oral tissues in a number of investigations. Grimsdottir
et al. (15) also studied the cytotoxic effect of orthodontic appliances in an
attempt to obtain a measure of tissue irritation caused by corrosion. Such data
do not allow derivation of an objective measure of copper irritancy; however,
release of metal ion in a simulated environment is highly dependent on presence
and concentration, and thus the (galvanic) interaction with other metals,
resulting in variable and unpredictable concentrations/cytotoxicity of individual
metal ions, e.g., copper, as most of the metal is protein bound.
Intrauterine Devices
Increases in systemic copper via parenteral entry from a contraceptive IUD can
lead to adverse effects reported: systemic nonspecific contact dermatitis and
immediate immunologic contact urticaria, even though the amounts liberated
from such a device are relatively low. Copper levels determined in intrauterine
fluids from women who had used the T-380 A device were 11.4 4.7 mg/mL
after six months; 11.54 7.0 mg/mL after one year and 6.24 1.5 mg/mL after
three years. Overall, concentrations over the entire period surveyed ranged from
3.9 to 19.1 mg/mL (16). It is inferred that the toxic effect of copper ions thus
released in the uterus (present in the form of complexes with proteins) are
responsible for cutaneous eruptions, although most of the reported cases appear
to belong to the category of nonimmunologic systemic contact dermatitis (16).
These investigations on the release of copper ion from alloys in the
physiological environment in vivo and in vitro and the potential biological
effects from exposure help to explain the cases of systemic irritative response
to IUDs and dental materials (17,18). On close examination, the immunologic
relevance of many of those reports is unclear.
INCIDENCE AND EPIDEMIOLOGY OF IRRITATION
DUE TO COPPER
Incidence of irritant contact dermatitis (ICD) is difficult to establish, as often
patients do not consult a doctor. ICD, especially of the hands, is reported to

Irritation Potential of Copper Compounds 101
be more common in women than men, possibly due to greater exposure
to irritants in ‘‘wet work’’ in the household; also, women are more likely
to consult with a doctor than men are. Studies in twins indicate that heredity
is a factor in susceptibility to irritants, but variability is too great for generalizations.
Atopic dermatitis seems to bring greater risk for ICD (18,19).
Based on 5839 dermatology patients patch tested by the North American
Contact Dermatitis Group, in which the role of occupational exposure to allergens
and irritants was evaluated, 19% were found to be occupationally related.
Of those, 60% were of allergic and 32% of irritant origin. The hands were the predominant
part of the body affected, 80% of thosedue to exposure to irritants (20).
For copper specifically, the aspects of epidemiology, prevalence, or
population studies cannot be addressed since, in contrast to other metals such
as nickel or chromium, reports of untoward reactions, systemic as well as
cutaneous, are extremely rare. The two geographical areas with the most complete
databases are the National Office for Occupational Health (Helsinki,
Finland) and the State of California. The figures emanating from these
sources are skewed in that they probably represent a small portion of the
actual frequency of disease due to inherent weaknesses in reporting systems.
PHARMACOLOGY OF COPPER
Beneficial as well as adverse health effects due to copper, an essential trace
element, are well characterized. Two pathological conditions stand out due
to their chronicity. Menkes’ syndrome is remarkable in that there is no
known cure and homocygots usually die early in life.
Wilson’s disease (WD) is an inherited copper metabolism disorder,
impairing biliary tract copper excretion that leads to excessive levels of the
element in tissue, particularly in the liver if left untreated. Left untreated,
such copper accumulation leads to hemolytic anemia, which over the years
can result in progressive hepatic failure and ultimately death (21). The characteristic,
brown ‘‘Fleisher rings’’ that develop in the eyes of Wilson’s disease
patients are caused by the deposition of metallic copper. However, WD
is very much treatable, if not cured, by penicillamine therapy and dietary
control. WD patients lead seemingly normal lives as long as they are on
medication and restrict copper intake.
Deficiency of copper is associated with characteristic integumentary
and skeletal abnormalities, defects in growth and development, and
abnormalities in sensory perception (22). Copper status of the organism is
reflected in ceruloplasmin levels. Plasma levels below 125 mg/dL are generally
considered as indicative of copper deficiency (23).
Menkes’ Syndrome
Albinism, the striking absence of pigmentation in the skin, hair, and eyes, is
characterized by the absence of the copper enzyme tyrosinase, which

102 Hosty´nek and Maibach
converts tyrosine to melanin in the melanocyte (24). Menkes’ kinky hair
syndrome, a hereditary defect in intestinal copper absorption that causes
retardation in growth, focal cerebral and cerebellar degeneration, and hair
to be abnormally sparse and brittle, becomes manifest in early infancy (25).
Afflicted infants have low levels of copper and ceruloplasmin, dying usually
within the first year of life. Although copper absorption and reabsorption
are impaired, tissue copper levels of many epithelial tissues, including the
skin fibroblasts, are elevated, and an increased production of metallothionein,
the cysteine-rich protein that binds copper in cells, appears to be the
cause of such accumulation. The biochemical defect underlying Menkes’
syndrome however is largely unknown.
Copper as Antimicrobial
Copper itself proved highly antimicrobial in plumbing and in lab tests with
several bacterial strains and some viruses.
A chlorophyllin copper complex, derived from chlorophyll by replacing
the chelated magnesium with copper, has anti-inflammatory and antimicrobial
properties, as well as a marked stimulating effect on epithelial cell
growth rates and cell regeneration. First established in tissue culture studies,
these findings were confirmed clinically through wound healing and deodorizing
characteristics observed in animal and man (26). Administered orally,
chlorophyllin copper complex is classified as a safe and effective internal
deodorant by the U.S. Food and Drug Administration (27).
Transdermal Anti-inflammatory Action of Copper
Exogenous copper has demonstrable anti-inflammatory effect, as several
copper complexes like Cupralene, Dicuprene, Alcuprin, or Permalon are
successfully employed in treating human arthritis (28–31). The potential
for copper’s activity as an anti-inflammatory agent by transdermal delivery
is subject to controversy, however. This is because scientific studies designed
to demonstrate therapeutic benefits for arthritic conditions through dermal
contact with metallic copper so far have been inadequate. Quantitative data
for percutaneous penetration of copper’s putative oxidation products, which
may be generated in contact of the metal with skin in humans, are still
outstanding. One missing, important factor for a convincing case of such
potential benefits is the deficiency in systematic and adequate scientific
research into the penetration of copper through human skin in any of its
forms, as polar mineral salts or as the more lipophilic complexes, and thus
a lack of solid scientific data documenting the therapeutic value of transdermal
copper delivery. It can be safely assumed that endogenous copper has
natural anti-inflammatory activity, and that such activity may also be reinforced
by exogenous copper. In a review of anti-inflammatory activity of
exogenous copper, Milanino et al. (32) concluded that copper, indeed, is active

Irritation Potential of Copper Compounds 103
as an acute anti-inflammatory agent irrespective of chemical form, including
inorganic copper salts. That there is a direct connection between copper and
rheumatoid arthritis is supported by the fact that low molecular weight
copper concentrations in plasma and synovial fluids increase in response to
the disease, and when such increases are induced further by administration
of exogenous copper, they are observed to have a definite anti-inflammatory
effect in both laboratory animals and humans.
COPPER IRRITANCY IN SKIN AND MUCOSA
In Vivo Assays
Kinetics and specificity of nickel hypersensitivity were assessed by Siller and
Seymour in mice presensitized with nickel sulfate and challenged with Cu (II)
sulfate, chromic chloride, cobaltous chloride, nickel chloride, and nickel sulfate.
The challenge concentration for the metal salts was 0.0152 M, and for
Cu (II) sulfate, 0.003 M. A reaction occurred at 24 hours, resolving at
48 hours, consistent with an irritant reaction. Cu (II) sulfate was found to
be ‘‘profoundly more irritant than the other metals’’ (specific numbers not
given) (33).
The biocompatibility and metal release were investigated in vivo
through implantation of representative specimen alloys in rats, and in vitro
in a battery of cell culture tests (7). In addition, combinations of dissimilar
alloys were investigated in relation to possible enhanced corrosion by galvanic
effects. Implantation and cytotoxicity tests on epithelial cells, macrophages,
and erythrocytes were performed, and the results compared. The severity of
tissue response in implantation tests corresponded to the nobleness of the casting
alloys joined to amalgam. The most severe reaction occurred in the tissue
in proximity of the LG-1 alloy, probably due to its high copper content. Similar
results were obtained in the in vitro macrophage test. All of the alloys
except the high-gold alloy (LM-Hard) had a toxic effect on epithelial cells.
The combination of the casting alloys with amalgam diminished such toxicity.
For the study, limit ratios of the metals used in the alloy were evaluated
in order to test the biological significance of the galvanic currents with
respect to these materials.
Implantation Test
Twenty-eight Wistar rats, weighing 250–300 g, were implanted subcutaneously
with the various alloy combinations for time periods of 7, 30, and
60 days (Table 1). Two rats in each group were allocated for each alloy combination.
Polystyrene implants serving as controls were left in 10 rats for the
same observation times. After the allotted time, the animals were sacrificed
with ether. The specimens including the surrounding tissues, submandibular
glands, liver, kidney, spleen, and part of the spinal cord were examined.

104 Hosty´nek and Maibach
Table 1 The Compositions of the Examined Alloys (wt%)
Alloy Au Pt Pd Ag Cu Sn Zn
LM-hard 76.9 1 1.4 9.6 11.1
LG-1 a
50.0 9.0 4.0 34.0 2.9
Micro 5.7 1.0 25.0 67.2 1.0
Midi 44.9 0.1 3.0 39.0 12 1.0
ANA 68 67.6 5 26.2 0.26
Revalloy 69.6 2.8 26.9 0.96
a Experimental alloy.
The connective tissue reactions to the implants were diagnosed as mild,
moderate, or severe on the basis of the degree of infiltrate, vascularity,
and fibrosis. In addition, special attention was paid to estimation of the
giant cells and foreign bodies in the tissues removed. Whenever foreign
bodies were histologically recognized, Energy dispersion X-ray (EDAX)
analysis was performed to reveal their constituents.
Polystyrene control: The response to polystyrene was an uncomplicated
repair of the surgical wound. Collagen fibers were present at day 7,
and a thin compact collagenous capsule enclosed the implant by day 30, followed
by an acellular capsule detectable by day 60.
LM-Hard gold alloy: At day 7, the tissue surrounding the implant
showed a moderate inflammatory cell infiltration and proliferating fibroblasts.
Giant cells and a well-defined capsule were detectable at day 30.
The capsule matured to a dense connective tissue membrane by day 60. Foreign
bodies were still detectable around the implants after 30 and 60 days.
EDAX showed the presence of Au, Cu, and Fe in these bodies.
LM-Hard/ANA 68 combination: The inflammatory response around
the implant was extensive by day 7. Granulation tissue formation was
delayed, and characterized by numerous macrophages and extensive capillary
proliferation. Foreign body aggregates found around the implant were
verified by EDAX to consist of Au, Ag, Hg, Cu, Sn, and Zn. A subacute
inflammation with prominent vascularity still persisted at day 30. Foreign
bodies were shown to contain Cu, Hg, Fe, Sn, and Zn. At day 60, the inflammation
had resolved into the mild stage, and a collagenous capsule
surrounded the implant.
Micro/ANA 68 combination: The initial reaction of the tissue against
the Micro/ANA 68 combination presented heavy inflammation due to granulocytes,
plasma cells, and macrophages. The reaction subsided by day 30,
but the vascularity still remained prominent. By day 60, the tissue outside
the fibrous capsule contained a few accumulations of lymphocytes. EDAX
analysis disclosed the presence of Au, Ag, Cu, Fe, Hg, Sn, and Zn in the
foreign bodies adjacent to the implants.

Irritation Potential of Copper Compounds 105
Midi/ANA 689 combination: After the seven-day observation period,
the Midi/ANA 68 implant had induced a strong cellular reaction with pronounced
vascularity. Macrophages were abundant. After 30 days, there was
a fibrous inflammatory region adjacent to the implant, contiguous with a
zone of granulation tissue composed mainly of fibroblasts, mononuclear
cells, and small blood vessels. Foreign bodies around the implant contained
Ag, Au, Cu, Hg, Pd, and Sn. On day 60, a capsule with well-oriented collagen
fibers existed, with only a few inflammatory cells present.
LG-1/ANA 68 combination: By day 60 there still was a subacute inflammatory
infiltration with high cellularity, plasma cells, and lymphocytes in
predominance. Giant cells, macrophages, and occasional granulocytes were
also detected. Some collagen was apparent, but it was poorly orientated.
At this stage, foreign bodies were seen containing Ag, Au, Cu, Hg, and Sn
in abundance.
Histopathology: None of the biopsies from different parenchymal
organs showed any morphological changes due to implants. Occasionally,
foreign bodies or blackish precipitates were present in liver, kidney, and
spleen. EDAX analysis showed them to contain calcium, chloride, sulfur,
silicon, potassium, and iron in varying proportions. In addition, occasional
copper particles were found in the kidney following the implantation of the
LG-1/ANA 68 combination. Also, a few particles containing Ag were
detected in the spleen of the same animals.
The results obtained in the investigations of alloy combinations
showed that when implanted in living tissue they caused reactions different
in character and intensity, which finally led to the formation of fibrous capsules.
The authors conclude that the differences in the severity of the
responses observed can, in most instances, be explained on the basis of electrochemical
reactions due to the different electrical potentials responsible
for the release of metal ions. In the present study, EDAX analysis showed
the presence of alloy elements in the surrounding tissue in every case. The
composition of the elements was not identical to that of the original alloys,
which indicates in situ corrosion, rather than particles dislodged from the
test alloy during the implantation procedure. The severity and duration of
the inflammatory reaction around the implants fully corresponded to the
nobleness of the alloys, and, surprisingly, not to the suggested electric potential
difference generated between the combined alloys.
Using EDAX analyses, the foreign bodies adjacent to the gold (LM-
Hard) implant were always shown to contain both gold and copper, thus
suggesting that gold may be complexed to copper within cells, or evoking
a copper-like biological response that is also causing localized accumulation
of copper. Whether such a possible gold–copper complex is related to the
adverse effects of gold or is a normal pathway in gold metabolism is not
known. The finding that capsules around the amalgam implants contained
mercury and tin particles are in agreement with previous observations (34).

106 Hosty´nek and Maibach
The extensive reaction to the LG-1/ANA 68 combination is apparently
related to the release of copper from the LG-1 alloy. In vitro studies
on binary Cu–Pd alloys have shown that a preferential dissolution of Cu
is followed by an enrichment of Pd on the alloy surface (35). Amalgam, on
the other hand, corrodes continuously. The high copper content of LG-1
(34%) accounts for a continuous copper release with a slow rate of Pd
enrichment, thus maintaining a persistent inflammation with high cellularity
adjacent to the implant. These findings are consistent with recent reports
dealing with tissue response to Ag–Pd–Cu–Au I alloys and pure copper
implants (36,37).
The abundance of macrophages and copper around the LG-1/ANA 68
implants supports the results of McNamara and Williams (37) who showed
that the pigmented material found in connection with Cu implants was composed
of Cu-containing macrophages. The cells had absorbed large amounts
of copper and remained damaged in the area, attracting more macrophages
to these sites. Cu particles could seldom be found in the liver after implantation
of the LG-1/ANA combination. This is contradictory to the findings of
Yli-Urpo and Parvinen (38), who always found elevated levels of Cu and Hg
in the liver and kidney after implantation of different alloy combinations.
This discrepancy can be explained by the different methods used. The disadvantage
of EDAX analysis is that only the surface of the specimen to a depth
of 2–3 mm can be analyzed.
Agarose Overlay Test
The effects of alloys and their combinations on cultured human epithelial
cells were examined. The cytotoxic effect of the test alloy was evaluated
by measuring the zone of cell lysis around the alloy.
Midi produced the most prominent cytotoxicity, whereas LM-Hard
had no effect. All of the alloy combinations were less cytotoxic than the constituent
alloys when tested separately. The diminishing cytotoxicity was
most prominent with the combination of Midi/ANA 68.
The reaction between the alloy and the culture medium can result in the
leakage of metal ions from the alloy into the culture medium, while the cells
themselves have no detectable effect on the corrosion process. LG-l, Micro,
Midi, and ANA 68 alloys showed a marked cytotoxic activity in the agarose
overlay test. The release of copper could be the major factor responsible for
the observed rapid cytotoxic effect of Midi. The minor degree of cytolysis
caused by LG-1, despite its high copper content, might be due to the preferential
release of the least noble metal, zinc, thus retarding the release of the
more noble constituents, copper included. The equal degree of cytolysis
caused by Micro and ANA 68 (both containing equal amounts of silver) substantiates
the concept of the role of silver as a cytotoxic agent. Surprisingly,
the degree of cytolysis diminished when the casting alloys were combined
with ANA 68. This is probably due to the electrochemical passivation, which

Irritation Potential of Copper Compounds 107
was most pronounced in the Midi/ANA 68 combination, and least in the
LG-l/ANA 68 combination.
Erythrocyte Lysis Assay
When the hemolytic activities of the alloys were tested, Midi, Revalloy, and
LM-Hard were shown to possess a slight hemolytic activity. Microscopy of
the cell pellet did not show any hemagglutination, which otherwise might
cause low hemolysis values.
Incubation of Midi, Revalloy, and LM-Hard with erythrocytes resulted
in a slight degree of hemolysis. The mechanisms by which the metal particles
produce their biological effects are not known in detail. It has been proposed
that interaction between the erythrocyte membrane and the particles would
be the most important factor in hemolysis (29). Additional mechanisms conferring
the hemolytic activity are the chemical nature at the metal surfaces,
particle size, and their surface charge. Since LG-1 did not show any hemolytic
activity, cytotoxicity cannot be attributed to copper release. Further studies
are needed, however, in order to elaborate on the elements and membrane
components involved in the hemolytic mechanisms of dental alloys.
Toxicity Test Using Murine Macrophages
Latex and Revalloy particles are phagocytized faster than the other alloy particles.
In the cultures of macrophages, which had been in contact with LG-1, a
phagocytosis rate of only 25% was detectable, as compared to 80% due to
Revalloy. The number of alloy particles phagocytized per macrophage was
significantly lower than that of the Latex particles. The proportion of nonviable
macrophages after exposure to the alloys, except Microalloy, was slight.
More pronounced cellular damage with pyknosis and vacuolization appeared
after exposure to Microalloy. No difference in toxicity was observed after one
day compared to one-hour exposure to the alloys except for LG-1, which
showed cell damage comparable to that due to Microalloy. A considerable
amount of lactic dehydrogenase (LDH) was released by Microalloy. In contrast,
little LDH release occurred when the cultures were exposed to Revalloy
or latex particles.
Solubility of particulate alloys into the macrophage culture medium: The
concentration of zinc in medium from alloy cultures was higher than in
the controls. The solubility of Zn was most prominent from Midi alloy.
In addition, copper release from LG-1 and Midi alloys was found. No
release of Au, Ag, or Sn was detected in any of the culture media (Table 2).
The corrosion of metal implants in human and mammalian organisms
(due to body fluids) may lead to local reactions in the surrounding tissues.
The tissue response will depend on the corrosive behavior of the metal, the
rate of release of metal ions, and their physiological activity. Since each
element will be released at a different rate from a complex alloy and many

108 Hosty´nek and Maibach
Table 2 Soluble Metals Concentration in Macrophage Culture After One Hour
Particulate alloy Zn SD (mg/mL) Cu SD (mg/mL)
LM-hard 0.62 0.15 0.21 0.10
LG-1 0.71 0.09 0.71 0.30
Micro 0.70 0.14 0.14 0.04
Midi 1.45 0.61 0.52 0.44
Revalloy 0.83 0.21 0.13 0.17
Control 0.56 0.13 0.17 0.10
have a different mechanism of toxicity, it is difficult to establish the biocompatibility
of the alloy using a single test method. A combination of test
methods was used in an attempt to assess the behavior of a variety of complex
dental alloys in different bioenvironments.
Amalgam particles were phagocytized faster than the other alloy particles.
This might be due to the differences in particle size. The present results
indicated that particulate Microalloy was the most toxic of the alloys tested,
whereas particulate Revalloy was well tolerated by the cells. Analyses of the
soluble elements in alloys revealed only relative low concentrations of copper
and zinc. Gold, silver, and tin were not detectable in any of the supernatant
determined from the experiments described. It seems that the alloys are not
sufficiently soluble in tissue culture medium for their effects to be exerted
with extracellular toxic levels. These findings are in agreement with previous
reports where no definite correlations could be found between the solubility
of the particles and toxicity. Thus, it seems more probable that the alloys
exert their toxic effects directly intracellularly after being phagocytized.
Copper has been shown to cause degenerative changes in macrophage
morphology, which could explain the increased LDH values due to LG-1
and Micro, despite only mild and moderate changes in cell morphology.
Comparison of the different tests: Some correlation was seen between
the in vivo implantation test and the in vitro macrophage test. This can
be explained by the central role of macrophages in the manifestation of
inflammation. Furthermore, macrophages are the first cells with which foreign
bodies come into contact in living tissue. The in vitro results obtained
from the agarose overlay test and the erythrocyte lysis test did not correlate
well with the in vivo results. The toxicity established by the agarose overlay
test would indicate the toxicity of soluble silver and copper rather than that
of the alloy in itself. In hemolysis, on the other hand, interaction of the alloy
constituents with biomembranes is one of the likely mechanisms involved in
the toxicity of particulate alloys. Some evidence exists that materials that
have not been phagocytized but that come into contact with the cell surface
can cause macrophage destruction comparable to hemolytic activity. The
interpretation of the results obtained by the different test methods is difficult,
and the dynamic state of cells and their possible metabolic alterations due to

Irritation Potential of Copper Compounds 109
the implants are not fully understood. The behavior of alloys in a biological
environment and the precise effect of each constituent element in different
tests need to be studied more extensively.
The severity of tissue response in the implantation test corresponded
with the nobleness of the alloys combined with ANA 68. The most severe
reaction was seen in the area surrounding LG-l, probably due to its high
copper content. Similar results were obtained in the in vitro macrophage
test. The agarose overlay test showed a somewhat similar zone of lysis for
all the alloys except for LM-Hard. The combination of the alloys with
ANA 68 reduced the lytic zone, which could be accounted for by a surface
passivation of the alloy. LM-Hard, Midi, and Revalloy showed a slight
hemolytic activity. A poor correlation was established between the agarose
overlay, the erythrocyte hemolysis, and implantation tests.
In Vitro Assays
Schmalz et al. evaluated the suitability of a commercially available model
system based on a recombined coculture of human fibroblasts and human
epithelial cells for assessing mucosal irritancy of metals used in dentistry, as
no valid animal or in vitro model exists for this purpose (39). That model
had been introduced for evaluating the time-dependent irritancy of cosmetic
products, where cell viability and prostaglandin E2 (PGE2) release from
the cells were used as markers for the irritative potential of test materials. The
human fibroblast–keratinocyte cocultures were exposed to test specimens
fabricated from copper, zinc, palladium, nickel, tin, cobalt, indium of high
purity (99.98–99.99%), and from a dental ceramic. Cell survival rates decreased
after exposure to copper (14–25%), cobalt (60%), zinc (63%), indium
(85%), nickel (87%), and the nonoxidized/oxidized high noble cast alloy
(87%/90%) compared to untreated control cultures. Dental ceramic, palladium,
and tin did not influence cell viability. In parallel, the PGE2 release
was continuously monitored up to 24 hours using a competitive displacement
enzyme immunoassay. PGE2 release increased most highly in the cultures
exposed to copper (6–25-fold), cobalt (seven-fold), indium (four-fold), and
zinc (two-fold) compared to untreated control cultures. The PGE2 determination
proved to be a nondestructive method for continuous monitoring of cell
reactions in the same culture. The model used seems promising for evaluating
the time-dependent mucosal irritancy of dental cast alloys.
Cell viability of exposed cell cultures was determined by the MTT test
after 24 hours. Survival rates were calculated relative to values obtained in
untreated cultures. For PGE2 release, assay aliquots (100/mL) were taken
from exposed media and the amount of PGE2 released from treated and
untreated cell cultures was quantified against a standard curve of purified
PGE2, using a competitive displacement enzyme immunoassay. Threedimensional
fibroblast–keratinocyte cocultures were exposed to one high
noble dental cast alloy and various metals frequently found in cast alloys.

110 Hosty´nek and Maibach
Identical levels of cell viability were found in untreated control cultures and
in cultures exposed to a dental ceramic, which was used as a negative control
material. Pure copper was the most toxic metal tested. In copper-exposed cultures,
a time-dependent decrease of cell viability at a level of 14% to 25% of
untreated cell cultures was observed. Because of the demonstrated high
toxicity, copper was routinely included as a positive reference material in
all subsequent experiments evaluating the effects of other test materials.
Cobalt and zinc induced a moderate decrease of cell viability to a level of
about 60% of untreated cell cultures. Pure nickel, indium, and oxidized and
nonoxidized specimens of the high noble cast alloy, were weakly toxic. Similar
to the dental ceramic, no cytotoxicity was observed after exposure of the
cocultures to palladium and tin specimens. Survival rates after exposure to
copper, zinc, indium, cobalt, nickel, and the high noble alloy (oxidized and
nonoxidized) were significantly different from those of untreated control
cultures. Total amounts of PGE2 released from cell cultures exposed to test
materials and from untreated control cultures steadily increased during the
exposure period. The spontaneous PGE2 release from untreated tissues was
identical with values obtained from cultures exposed to specimens of the
dental ceramic and nontoxic metals. The amounts of PGE2 released after
exposure to copper were about 10-fold higher than those released from
untreated cultures after a 24-hour exposure. After 30-minute exposure to copper
specimens, significantly higher PGE2 levels were already found compared
to untreated controls. In contrast, no differences were found between the
PGE2 levels measured in media of untreated tissues and tissues treated with
all other test materials. In repeated experiments, the amounts of PGE2
released from cultures exposed to copper varied, being 6–25-fold higher than
those released spontaneously. Indium and cobalt in contrast produced
increases that were considerably lower than those elicited by copper in the
same experiments (4–7-fold). The induction of an increased PGE2 release
from human fibroblast–keratinocyte cocultures was inversely related to cell
viability measurements after exposure to copper. The dramatic effect of
copper on cell viability is in accordance with data from other in vitro and in
vivo studies (40,41). This is due to the oxidative potential of pure copper
and the toxicity of copper ions in vitro (42,43). As a consequence of copper
toxicity, the cell viability was reduced to about 15% to 25% of untreated control
cultures. The increases of PGE1 levels by factors of 2 (zinc) to 25 (copper)
are among the highest observed in vitro so far (44,45). The model system based
on a recombined coculture of human fibroblasts and human epithelial cells
seems promising for evaluation of the mucosal irritative potential of dental
materials; however, further studies, particularly on interexperimental variations,
are needed before it can be established as a routine test model candidate.
Cell viability as measure of cytotoxic potential in HaCaT cells
(a spontaneously immortalized human kertinocyte line) and, indirectly, of
irritancy in vivo, was determined on human keratinocytes in vitro by Brosin

Irritation Potential of Copper Compounds 111
et al. (46) for five metal salts. The endpoint used to assess cellular viability
was metabolism of the tetrazolium salt XTT [2,3-bis (2-methoxy-4-nitro-
5-sulfophenyl)-5-(phenylamino carbonyl)-2H-tetrazolium hydroxide]. The
metal salts showed the following rank order in cytotoxicity at an exposure
time of 24 hours: potassium bichromate > Cu (II) sulfate > cobalt chloride
and palladium chloride > nickel sulfate. The authors found an excellent correlation
to the rank order of the metals’ known irritative potency, as it was
determined in vivo for purposes of contact allergy screening by the ICDRG,
but recognized that such a test hardly applies to the complex pathomechanism
of skin irritation. As such, the presented XTT assay on HaCaT cells would be
well-suited for an initial screening of substances to establish a relative order of
irritancy as part of a battery of tests targeting different aspects of skin irritation.
This could be subsequently followed by irritation tests in humans.
CONCLUSIONS
Data on the dermal irritation by copper and its compounds is scant, and
its irritancy has not determined, e.g., in terms of an irritant dose ID50.
Irritancy of copper can only be comparatively characterized in relation
to other metal salts. A rank order for the irritancy of metal compounds
can be inferred from the patch test concentrations recommended as nonirritating
for the purpose of cutaneous allergy testing: potassium dichromate
0.5% in petrolatum; copper sulfate, cobalt chloride, and palladium chloride
ex equo: 1% in aqueous solution; and nickel sulfate: 5% in petrolatum.
With the exception of its mineral salts, copper (II) compounds (complexes,
soaps) exhibit low irritancy and several have been adapted as therapeutics
for epicutaneous applications as antiseptics or deodorants (e.g., the
chlorophyllin copper complex, gluconate, oleate, or citrate) or in
transdermal drugs (copper salicylate, copper phenylbutazone).
Because of the increasing need for reliable skin irritation tests and in
order to reduce the number of animal experiments, in vitro alternatives
have been developed. So far, in vitro studies show that different chemicals
induce irritant inflammatory responses, which vary considerably in the time
course of the response, and that there are differences in the components of
the inflammatory response to different irritants. Although no single test can
be considered as an indirect, though reliable, measure of skin irritation in
vivo, a battery of tests, each addressing a different aspect of such multifactorial
phenomena leading to skin irritation, may well be a critical step
preparatory to in vivo testing in humans.
Distinguishing between irritant and allergic contact dermatitis can be
challenging; thus, copper cross-reactivity/concomitant sensitization with
other transition metals and failure by practitioners to resort to patch testing
for resolution of questionable skin reactions, in many cases, leads to questionable
diagnosis of irritation.

112 Hosty´nek and Maibach
Cu (II) sulfate is clearly an irritant when applied in pet under occlusion
for 48 hours. However, there are no currently available data that allow us to
determine the threshold for induction of acute or cumulative irritancy
dermatitis for copper or any of its salts. Fortunately, the technology to
define this is readily available (cumulative irritancy testing). These are now
being generated in this laboratory.
ABBREVIATIONS
ACD allergic contact dermatitis
ARL adult rat lung
ELISA enzyme-linked immunoassay
ICD irritant contact dermatitis
ICU immunologic contact urticaria
IUD intrauterine device
LDH lactate dihydrogenase
MM mitochondrial membrane
MTT mitochondrial toxicity test
NICU nonimmunologic contact urticaria
SC stratum corneum
TEWL transepidermal water loss
TLV threshold limit value
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Acta Derm-Venereol (Stockh) 1997; 77:26–28.

7
Copper Hypersensitivity: Dermatologic
Aspects—Overview
Jurij J. Hosty´nek and Howard I. Maibach
Department of Dermatology, University of California at San Francisco
School of Medicine, San Francisco, California, U.S.A.
INTRODUCTION
Reports of immune reactions of both the immediate and delayed types due to
cutaneous or systemic exposure to copper have been reviewed, in the endeavor
to draw a comprehensive profile of the immunogenic potential of that metal
and its compounds. Also the metal’s immunotoxic potential is briefly reviewed.
In principle, as noted for other transition metals, the electropositive
copper ion is potentially immunogenic due to its ability to diffuse through
biological membranes to form complexes in contact with tissue protein.
Based on predictive guinea pig test and the local lymph node assay,
copper has a low sensitization potential. Reports of immune reactions
to copper include immunologic contact urticaria (ICU), allergic contact dermatitis,
systemic allergic reactions, and contact stomatitis, but considering
the widespread use of copper intrauterine devices (IUDs) and the importance
of copper in coinage, items of personal adornment, and industry, unambiguous
reports of sensitization to the metal are extremely rare, and even fewer
are the cases that appear clinically relevant.
This chapter, in part, was reprinted from Hosty´nek JJ, Maibach HI. Copper hypersensitivity:
dermatological aspects—an overview. Rev Environ Health 2003: 18:153–185, with permission.
115

116 Hosty´nek and Maibach
Reports of immune reactions to copper mainly describe systemic exposure
as cause to IUD, and to prosthetic materials in dentistry, implicitly
excluding induction of hypersensitivity from contact with the skin as a risk
factor. We provide a diagnostic algorithm that might clarify the frequency
of copper hypersensitivity.
Objective
Intent of the overview is to establish a synopsis of dermatologic immune
reactions ascribed to copper exposure, and to examine the criteria applied
in such diagnosis, since not always has such causation been demonstrated
unequivocally. The review discusses metallurgy of copper, predictive and
diagnostic tests and describes types of immune reactions and the potential
for the copper ion to act as sensitizer, followed by critical examination of
literature reports applying strict diagnostic criteria, with consideration given
to a number of confounding factors that may have led earlier investigators to
erroneous interpretation of signs, symptoms, or test results.
The last decade has seen a marked expansion in interest in metalallergic
contact dermatitis—from a focus mainly on nickel and chromate
to currently gold, cobalt, palladium, and others. Case report methodology
now is much of the literature citations in this area. Here, we critically review
the citations and suggest diagnostic criteria that might clarify how often
copper hypersensitivity occurs in man.
The skin is a target organ and indicator for allergy. While the stratum
corneum (SC) is a partial barrier to the passive penetration of allergens, to
electrophilic, protein-reactive metals in particular, live tissue of the epidermis
and dermis actively process penetrants or systemically absorbed
allergens that reach it. Such immune reactions to chemicals in the skin are
broadly categorized into two distinct classes:
1. Allergic contact dermatitis (ACD) or delayed-type reactions
mediated by allergen-specific T lymphocytes. It expresses as a wide
range of cutaneous eruptions upon (a second) dermal contact or
systemic exposure to haptens in individuals with preformed cellular
immunity (type IV allergic reactions).
2. ICU or immediate-type hypersensitivity, which involves IgE antibody.
The latter most notably results in respiratory allergy, but
can also manifest in separate stages collectively described as ‘‘contact
urticaria syndrome’’ (1), local or generalized urticaria, urticaria with
extracutaneous reactions such as asthma, rhinoconjunctivitis, and
gastrointestinal involvement, and ultimately anaphylaxis (type I
reactions).
Copper has been alleged to sensitize de novo on systemic exposure following
inhalation or implantation. The resulting dermatosis thus induced is

Copper Hypersensitivity 117
described as systemic contact dermatitis or urticaria (2–5). Copper complexes
are also known to elicit skin reactions upon systemic challenge in
the previously sensitized organism (6).
METALLURGY OF COPPER AND ITS ALLOYS,
AND ITS ROLE AS SENSITIZER
Dissimilar metals, combined in alloys for the fabrication of medical devices
such as dental materials, evoke currents in electrolytic media such as saliva
and degrade, resulting in a steady release of metal ions. In immediate proximity
of dental restorations or IUDs, this can lead to adverse (intraoral or
intrauterine) reactions such as lichenoid lesions of the oral or genital mucosa.
Beyond local effects at the implant site, ions can be transported into distal
tissues such as the skin, giving rise to pathological processes such as manifest
allergic reactions. Among the metals that commonly form allergenic
ions are nickel, cobalt, chromium, and mercury. Exposure type, duration,
and environmental conditions (sweat, oxygen supply) in proximity of the
metal are critical for mobilization of ions leading to induction or elicitation
of immune reactions. As most articles of common human contact are alloys
and not made of the pure metal itself, electrochemical interaction between
components are significant for the release of allergenic ions potentially leading
to immune reactions (7).
Reports of copper as immunogen are few, and rarely could the clinical
relevance of copper sensitivity be demonstrated with certainty. Consequently,
the question as to incidence or prevalence of copper sensitivity
among the general population is moot, the number of cases too low to
express as percentages. Nevertheless, two characteristics of copper in contact
with tissues put the metal into a category that renders appropriate a
discussion of its role in inducing reactions in the immune system.
Copper belongs to the family of electrophilic transition metals, which
makes the copper ion highly protein reactive, i.e., likely to be haptenized,
thus recognizable by the immune system as non-self or foreign.
Although belonging to the nobler metals highly resistant to corrosion
(oxidation, dissolution), in the physiological environment (IUD, dental
materials, implants) or in contact with skin exudates, elemental copper is
converted to diffusible forms that can penetrate biological membranes.
This latter factor merits detailed discussion, also to lay the groundwork
for demonstrating how copper and other metals eventually become
biologically available from contact with endothelial and epithelial barriers.
The oxidation of copper (0) in body fluids has been investigated as a
factor that may determine induction or elicitation of immune reactions.
Release of metal ions experimentally determined in synthetic body fluids
may not adequately mimic the degree of corrosion (oxidation, release) as it
occurs in contact with live skin or in the physiological environment,

118 Hosty´nek and Maibach
however. This is due to the fact that composition of such media used for
routine experimentation lacks important components that, in contact of
foreign materials with a living organism, determine the nature of reaction
product, the rate of reaction, and thus the path of diffusion of the end products
through biological barriers.
These formulas appear to omit important factors, for instance those
present in skin exudates, which can play a determining role in metal oxidation:
proteins, and, most importantly, free fatty acids in the sebum (8–13).
Together with metal ions the latter are likely to form lipophilic soaps,
presumably diffusible via the intercellular lipid matrix of the SC. Evidence
for skin diffusivity in a model experiment was obtained by in vivo application
of copper oleate over 24 hours on human back skin. Urinary copper levels
were subsequently seen to increase significantly over several days (14). While
it is a good indication of facilitated permeation, that result in itself does not
indicate the actual path followed by the permeant, however. Evidence for an
actual path of diffusion was obtained in a different experiment; localization of
copper in the intercellular spaces was made visible through electron microscopy
following application of copper acetate on human skin (15).
Human plasma or serum are the most corrosive physiological media
and can play a decisive role on the path towards systemic immunization.
Comparative tests simulating corrosion of implant metals in vitro demonstrated
that the electrochemical process of oxidation in the presence of
enzymes, proteins, and other components of actual serum is accelerated in
comparison to standard simulating media (16). Corrosion testing of
implants thus becomes more relevant for in vivo conditions when it is conducted
in a proteinaceous medium (whole blood, serum, saliva) (16,17).
The present synopsis of hypersensitivity cases arising from contact
with copper amply, albeit indirectly, confirms the diffusivity of copper derivatives
through biological barriers. In addition, copper derivatives (abietate,
naphthenate, oleate, sulfate, 8-quinolinolate) used as pesticides are reported
to act as irritants when coming in contact with the skin—evidence for their
diffusion beyond the SC, reaching the live strata of the skin (18–20).
The practice of using copper compounds, including metallic copper, as
patch test materials for diagnostic purposes in dermatology also is based on
empirical evidence gathered for their diffusion to reach the live strata of the
epidermis when applied under occlusion.
Finally, conversion of copper metal to diffusible compounds has been
demonstrated in our laboratory in a semiquantitative manner (unpublished
data). The SC of human volunteers was analyzed in depth for copper
content following application of finely distributed metal on the skin under
semiocclusive conditions. After application of the metal as micronized powder
on the volar forearm for periods up to 72 hours, inductively coupled
plasma mass spectroscopy analysis of sequential tape strips showed that
the gradients of copper distribution profiles increased proportionally with

Copper Hypersensitivity 119
occlusion time, from 24 to 72 hours, rising to 10 ppm after the longest period,
significantly above the initial background level of 2 ppm.
PREDICTIVE IMMUNOLOGY TEST RESULTS FOR COPPER
Thus far, copper has been tested for sensitization potential in two predictive
tests: the guinea pig maximization test (GPMT), a standard method used as
predictor of skin sensitization potential, and in the local lymph node assay
(LLNA) (21,22).
In the GPMT, on 20 guinea pigs Boman et al. (23) noted two positive
reactions at 24 hours and seven at 48 hours after using 1% copper sulfate
pentahydrate in pet. Karlberg et al. (24) later found no difference between
copper-exposed and control animals at 1% to 0.1% CuSO4 in pet. Basketter
et al. (25,26) obtained a 0% response in the same test, but later in the LLNA
the result was positive.
In the LLNA adapted to test for allergenicity of metal salts also, under
modified conditions, cupric ion significantly increased lymph node cell proliferation.
Testing of cupric ion as the chloride in dimethyl sulfoxide at 1%,
2.5%, and 5% concentrations showed significant increases in lymph node
cells proliferation, with ratios of test to control lymphocyte proliferation
of 8.1, 13.8, and 13.6, respectively. Also, mice could be sensitized in the
LLNA by application of copper (II) sulfate (27,28). When the National Toxicology
Program Interagency Center for the Evaluation of Alternative
Toxicological Methods tested cuprous chloride in the LLNA, that copper
salt was also found to increase lymph node cells proliferation, resulting in
a positive test reading (29).
DIAGNOSTIC TESTS FOR HYPERSENSITIVITY
A differential diagnosis of chemically induced urticaria (ICU), immediatetype
irritant (non-immunologic contact urticaria) and ACD is sometimes
difficult, particularly when dealing with strong irritants. In simplest terms,
it is mainly based on concentration of the xenobiotic (agent) necessary to
induce a skin reaction, and on the time course of reaction.
The Open Test
The material is applied to intact skin or slightly dermatotic skin, with wheal and
flare developing in minutes, a positive indication of or ICU (see above).
The SPT for Immediate-Type Allergy (Contact Urticaria)
Sensitization is defined as a positive skin prick test (SPT) response with or
without clinical symptomatology. One drop (20 mL) of putative allergen in
an appropriate solvent (e.g., propylene glycol), vehicle (negative control),

120 Hosty´nek and Maibach
and histamine in physiological saline (positive control) is placed on three
separate sites on the volar aspect of the forearm. Using a sterile prick device
inserted through the drop, the underlying superficial epidermis is gently
pricked. One needle is used per skin site and discarded. Immediately after
pricking, each skin site is blotted dry. After 15 to 30 minutes the skin sites
are evaluated for wheal and flare response. An edematous reaction (wheal)
of at least 3 mm in diameter, surrounded by a flare, and at least half the
size of the histamine control is considered positive in the absence of such
a reaction in the vehicle control. SPT positives are retested to confirm the
response. Ultimately, diagnosis should be based on clinical history and
negative controls (30). Unlike with the open test, controls are mandatory.
Radioallergosorbent Test
Immediate allergic hypersensitivity can be diagnosed by radioallergosorbent
test (RAST), an in vitro immunologic procedure designed to detect specific
IgE antibodies in serum (31). Initially, a hapten–protein conjugate between
a reactive compound and human serum albumin (HAS) has to be synthesized
for the radioimmunoassay. The allergen (hapten–protein conjugate)
is coupled to a paper disc. IgE antibodies in a serum sample, which are specific
for the conjugate, bind to the conjugate epitopes on the disc, and the
portion of bound IgE is detected by 125 I-labeled anti-human IgE. Usually,
the results are expressed as a percentage of the total activity, the ratio
between the binding to the hapten-HAS disc and a disc onto which HAS
had been coupled and run in the same experiment.
The RAST Inhibition Test
Also cross-reactivity of various haptens can be determined by the RAST
method. Serial dilutions of conjugate are allowed to react with an individual’s
serum. The mixture is then used for RAST determination. Degree
of reduction of the serum RAST values after absorption are expressed as
percent inhibition (32).
The Patch Test for Delayed-Type Allergy (ACD)
The key diagnostic tool for ACD is patch testing. Objective is to reproduce
the skin reaction to a suspected allergen under controlled conditions,
by dosing the substance (or a standard series of allergens) in a suitable
vehicle at a nonirritant concentration on adhesive tape and placing it on
the skin. Penetration through the SC is promoted by airtight occlusion.
The test is left on for at least 48 hours. Some agents elicit reactions only after
a substantial delay (late phase reactions), such as copper or gold, possibly
due to their known anti-inflammatory activity (33–39). Compounds of poor
skin diffusivity such as transition metal salts may produce reactions with
considerable delays also (36,37).

Copper Hypersensitivity 121
It is suggested that in order to resolve doubtful cases a more differentiated
approach using dilution series is advisable for diagnostic purposes.
This would include using a dilution series rather than the standard practice
of applying the single 1% or 2% copper sulfate solution for patch test assessment
of allergic response to the metal salt.
Detection of allergens by patch testing with salts dissolved in water, dispersed
in petrolatum, or in their elemental form, and subsequent removal of
the allergen resulting in clinical improvement are the simplest and most direct
connections between cause and effect. As described in greater detail below, a
confounding factor in etiology and diagnosis for a number of transition
elements, and particularly in the case of copper, is the well-documented
cross-reactivity with other metal ions, primarily nickel, but also palladium.
These are reactions occurring when haptens of similar size and electron shell
configuration are transferred to the same carrier protein. In fact, in the
majority of copper sensitivity cases reported, the patients, when tested for
multiple metal allergies, were positive to two or more metals, nickel being
the most frequent one (40). That is an allergen that can induce clinical
manifestations in even minute amounts, and is ubiquitous in the normal
environment (41). Thus, false-positive reactions to copper may be due to presence
of trace contaminants in the putative cause for allergy, e.g., the IUD, or
even in the diagnostic test material, e.g., the metal disc (also see later).
Because of the inherent irritancy of copper sulfate under patch test
occlusion, and the relatively small number of normal volunteer controls used,
we estimate that the nonirritating dose for diagnostic testing approximates
1% to 2% in petrolatum. For verification of copper allergic hypersensitivity,
application of 1% copper sulfate in water or petrolatum is recommended by
the International Contact Dermatitis Research Group, or of an occluded
copper (metal) disc over 2 to 4 days. Petrolatum is the recommended vehicle
for copper sulfate, although uniform distribution of the crystalline salt is
problematic and poor penetration from petrolatum makes that choice less
than ideal. Any positive reactions warrant further evaluation to ascertain
clinical relevance (42).
One alternative method advocates the use of metals in the elemental state
for diagnostic skin tests, and several authors have used copper discs or currency
for patch testing (33,43–53). The diagnostic value of this approach is
put in question by one investigation, however, where metallic copper was
immersed in synthetic sweat to analyze for metal release. Over 24 hours, the
final copper concentration was 0.01%, considered by the authors to be too
low to elicit a reaction except in highly sensitized individuals (54).
Role of Vehicle in Patch Testing
In choosing a vehicle for percutaneous penetration, a factor for consideration
is the effect it will have on the skin membrane and thus its barrier

122 Hosty´nek and Maibach
properties, since the solvent of a xenobiotic (metal) can significantly influence
its diffusivity and thus bioavailability. Petrolatum, for instance, is a
poor solvent for metal salts because the permeant remains suspended as fine
particles affording less than ideal uniformity in skin contact, but on the
other hand it has an occlusive effect that would increase skin hydration and
thus promote diffusion of a hydrophilic compound. Another solvent that
enhances penetration is dimethylsulfoxide (DMSO) (see later for mercury
chloride). As an instance, Sharata and Burnette point to dimethyl formamide
and dimethyl acetamide associated with DMSO, which cause swelling
of basal SC cells and disrupt the normal keratin pattern. They located the
electron-dense metal ions mercury and nickel in the intercellular spaces
and corneocytes, whereas in control membranes those metals were seen
almost exclusively in the intercellular space. Thus, certain solvents may
modify intercellular solute diffusion to include the transcellular path (55).
How the nature of the vehicle can either influence the rate of release of a
compound or modify the barrier properties, thus determining the level of
percutaneous absorption of xenobiotics, is illustrated by further examples
from the literature. An instance of practical importance is the choice of
vehicle in standard diagnostic skin patch testing for sensitization, with the
aim of optimum release of allergen into the viable epidermis while avoiding
allergic or irritant contact dermatitis, leading to false-positive reactions
caused by the vehicle itself.
The enhancing effect on skin penetration by DMSO was demonstrated
by experimental results on guinea pigs in vivo. The use of neat DMSO as
vehicle for 0.239 M HgCl2 significantly increased the skin penetration of
the compound relative to water as vehicle, and thereby the percutaneous
toxicity (56). Mortality for the test animals at three weeks post-treatment
increased from 20% with water to 80% with DMSO. Permeability coefficients
for mercury in DMSO averaged 2.7 10 3 –4 10 3 cm/hr for the first
five hours, as compared to 0.44 10 3 –1.47 10 3 cm/hr when the carrier
was water.
Poorer penetration of salts formulated in petrolatum was demonstrated
repeatedly. Fullerton et al. (57) explored the effect of water as vehicle
for NiCl2 and of petrolatum for both NiCl2 and NiSO4 at l.32 mg Ni/mL
through in vitro experiments with full-thickness human skin.
Petrolatum was the poorest vehicle for NiC12 as less than 1 mg Ni/cm 2
reached the receptor in 70 to 98 hours compared to 12.6 mg Ni/cm 2 in water.
For NiSO4 in petrolatum, a poorer penetrant than NiCl2, no detectable
nickel reached the receptor phase over 163 hours.
The permeability coefficients for 2.4% zinc chloride through human
skin in vitro were compared when applied from petrolatum and from a
hydrogel over 72-hour periods. The permeability coefficient from petrolatum
was 0.082 10 4 cm/hr. Applied from the hydrogel base, the permeability
coefficient was 0.29 10 4 cm/hr, more than three times higher (58,59).

Copper Hypersensitivity 123
One method for determining the optimal solvent in diagnostic skin
patch testing of allergens is its application on hypersensitive patients and
recording the ratio of positive elicitation reactions. Thereby the solvent is
identified, which better promotes diffusion of the xenobiotic. The reaction
threshold to nickel sulfate was tested in 53 sensitized patients in both water
and petrolatum at equal concentrations (390 ppm) (60): in petrolatum three
patients were positive, and five to the aqueous solution, leading to the conclusion
that the mean reaction threshold for nickel sulfate in water is lower
(0.43%) than in petrolatum (51%) due to better diffusivity in the former. Also
the irritation potential of a chemical can be assessed by application in different
solvents in dermatological diagnostics aiming to minimize the chemical’s
potential for irritation and thus false-positive reactions. The irritant reactivity
of nickel salts in petrolatum was found to be greater than in that of
water (61). These two concordant examples from dermatological practice serve
to illustrate the differentiated promotion of diffusivity by different vehicles.
From experience in dermatological practice, particularly in consideration
of the clinical picture emerging from the few cases that document
copper allergy, and because of the complexity of the irritant dermatitis
syndrome, adhering to the criteria set out in the Operational Definition of
ACD is recommended when a definition of clinical relevance is sought (42,62).
TEST CONCENTRATIONS FOR COPPER ACD
Since relatively few dermatotoxicological investigations have researched
copper’s characteristics as allergen, no definite value has been assigned as
to copper sulfate’s threshold-inducing sensitization, nor is an optimal
concentration defined that would reliably elicit reactions in the sensitized
organism. Thus, the patch test doses vary: 1% aq. (Gruppo Italiano
di Ricerca Dermatiti da Contatto); 1% to 2% pet.; in a dental screening tray
concentrations include 1% aq. and 2% aq. (63,64).
IMMUNOGENIC POTENTIAL OF COPPER
Systemic ACD
Systemically induced allergic disease that can be caused by T-cell-mediated
reactions to metals (copper) (65), potentially occur when copper or coppercontaining
alloy materials used in IUDs, implants in replacement surgery or
orthodontic appliances, are oxidized with release of free copper ions. These
are absorbed through the epithelia and carried to the skin and the mucosa
via blood and the lymphatic circulation. There the allergen is intercepted
by antigen-presenting cells and recognized by T cells that migrate to the
lymph nodes with blastic transformation, proliferation of cytotoxic lymphocytes,
and production of cytokines. These in turn recall neutrophils and
eosinophils to the reaction site, cause capillary dilation and increased

124 Hosty´nek and Maibach
permeability, resulting in cutaneous inflammation appearing as wheal and
flare (Menne, 1996 membrane); lichen planus and asymptomatic contact
hypersensitivity (dental alloy contact dermatitis) are increasingly being linked
with oral exposure to materials used in dental fillings, orthodontic prostheses,
cements and components of dentures, bridges, bands, and wires. Such reactions
can be either immunologic contact stomatitis or systemic anaphylactic
stomatitis (type I reactions), or delayed contact stomatitis (type II). In a few
instances, copper was implicated as possible cause for the latter, as copper is
commonly a part of alloys used in dental materials (Table 1) (33,66,67). In
a study investigating the release of copper from a selection of orthodontic
appliances in organic and inorganic solutions made up to different pH values
to imitate the oral environment, Stoffolani et al. found that the levels of metal
mobilized were well below those ingested with a normal daily diet. From that
of result they concluded that the quantities released should be of no concern.
The relevance of that conclusion, particularly for purposes of immunology,
invite further discussion, however, to be pursued elsewhere (68).
A study of professionals (dental technicians, orthodontists and their
assistants) involved in making and handling such materials reveals that in
Table 1 Copper (0) and Nickel (0) Content (wt%) of Dental Materials
Alloy Cu, wt%
Ni, wt%
(other metals) Manufacturer
Copper (0) 100 N/R AB JS Sjöding, Kista, Sweden
JSC—gold alloy 11.5 N/R (Au, Ag,
Pt, Zn, Ir)
AB JS Sjöding, Kista, Sweden
Degussa Training 87.5 N/R
Hereaus Kultzer GmbH, Hanau,
Metal
(Zn, Sn, Co) Germany
Trindium 87.0 1.0
Trindium Corp. of America,
(Al, Mn) Los Angeles, California, U.S.A.
Duracast MS 81.6 4.1
Duracast Inc., Brasilia,
(Al, Fe, Mn) Sao Paulo, Brazil
Goldent 76.0 0.5
Goldent Inc., Brasilia,
(Al, Zn, Mn) Sao Paulo, Brazil
Modulay 77.0 N/R
J. F. Jelenko, Armenac, NewYork,
(Au, Ag, Pd) U.S.A.
ANA 68 5.0 N/R
AB Nordiska Affineriet ANA,
(Ag, Sn, Hg) Helsingborg, Sweden
Dispersalloy TM
12.4 N/R
J & J Dental Products Co.,
(Ag, Sn, Zn) Tallaght, Dublin, Ireland
Valiant TM
20.0 N/R
L.D. Caulk Co., Dentsply Intl,
(Ag, Sn, Pd) Inc., Milford, Delaware (U.S.A.)
Gallium alloy GF 15.0 N/R
Tokuriki Honten Co. Ltd., Tokyo,
(Ag, Sn, Pd) Japan
Abbreviation: N/R, none reported.

Copper Hypersensitivity 125
handling dental devices they also run the risk of developing hypersensitivity
to allergenic materials, metals among them, as in one study 40% of orthodontists
and 43% of dental assistants reported work-related skin problems (69).
Copper Intrauterine Devices
Copper metal in contact with biological substrates (as in IUDs) is highly
reactive and releases free copper ions. Release in vivo was determined
at 0.71 I-1 micro mol/day (45 I-1 micro g) from a surface area of 200 mm 2 and
1.29 I-1 micro mol/day (82 mg) in a culture medium in vitro (70).
After it was discovered that copper metal placed in the uterus of animals
had a contraceptive effect (71), the principle was applied to humans: a
plastic T-shaped device with copper wire or a copper sleeve was introduced
as a pharmacologic agent and became widely used as an IUD to regulate
fertility. Research suggests that copper prevents fertilization rather than
implantation (72).
Reports of untoward reactions by women using the IUD (generalized
eczema, edema) sometimes mention that the condition worsens during the
perimenstrual period of the cycle, and patients often test positive to patches
of copper as metal or as the sulfate (48). Upon removal of the device
patients usually experienced complete remission.
Dual Immune Response to Copper
While organic compounds infrequently cause both types of reactions, dual
immune response appears more common for metals and metallic compounds.
Their reactivity towards protein results in a complete antigen that triggers
both IgE antibody production (type I) and cellular (T cell, type IV) immune
reactions. Immunogenic effects that result from exposure to metals can be
attributed to the same factors that determine their toxicological and biological
effects. Metal ions in general, and certainly those belonging to the transition
group of elements, such as copper, have an ionic radius too small to be
antigenic. Containing a partially filled d shell, these metals oxidize to highly
electropositive cations that can act as haptens interacting with tissue protein.
They form bonds that range from fully ionized to fully chelated complexes,
and have the ability to modify the native protein configuration. These are
recognized as non-self by hapten-specific T cells in the host immune
system (73), leading to allergic reactions of the two different types.
Copper is one of several metals causing more than one type hypersensitivity
presenting with multiple symptoms in allergic responses, in part
depending on type of exposure: immediate type, immunologic contact urticaria
sometimes associated with respiratory hypersensitivity, delayed-type
cutaneous hypersensitivity, systemic allergic reactions, and contact stomatitis
(2–4,33,45,47,51,66,74,75).
Concurrent occurrence of immediate- and delayed-type sensitivity has
also been observed in the same individual (50,76).

126 Hosty´nek and Maibach
It should be noted, however, that, overall, human case reports of
copper-induced immunologic reactions are rare.
The Major Factors with the Potential to Induce Copper Sensitivity
Dental Materials
Over the past 15 years, there has been considerable effort to replace the use
of traditional materials such as mercury in dental restorative work, and
dental casting alloys contain increasing amounts of copper. This use of various
substitute, metal-based materials has proceeded without the necessary
corollary knowledge of their irritant and allergenic potential, however.
In reports of contact stomatitis (contact allergy of the oral mucosa), no
reports of rechallenge have been published which would strengthen the
causal association with copper.
Tables 2–4 list the different types of allergic reactions associated with
exposure to copper.
Recommended Patch Test Procedure in Suspected Copper Allergy
of the Delayed Type
Establish clinical history (anamnesis) determining nature of contact
and physical form of putative allergen.
Physical examination of the patient.
Patch testing with 2% CuSO4 in petrolatum. In case of positive
outcome follow up with serial dilution patch testing (1%, 0.5%,
and 0.1%).
Repeat open application test (ROAT) or provocative use test
(PUT) with a dilution series of CuSO4: 2%, with at least 10 na€ve
control subjects to demonstrate that positive reaction is not irritant
in nature, then further at 1% and 0.5%. The substance is applied
once or twice daily for 14 to 28 days (107,108). A positive reaction
usually appears within four days, less frequently between five and
seven days. Delayed reactions have been noted in patch testing
with copper.
To confirm positive patch test reactions and identify false-negative
reactions on patch testing, intradermal tests may be considered
(109). Herbst et al. (110) provide the scientific background of intradermal
testing for ACD.
As an ‘‘alternative’’ predictive test for ACD, the local lymph node
assay was developed on mice for the detection of contact allergens (22). It
has been adapted to test for allergenicity of metal salts also. Under modified
conditions, cupric ion was seen to significantly increase lymph node cell
proliferation, as mice could be sensitized by application of copper (II)
sulfate (27,28).

Copper Hypersensitivity 127
Table 2 Immunologic Contact Urticaria Due to Copper
Visible signs and
symptoms Diagnostic test Challenge reaction CR References
Cases Cohort/etiology
1 Dental Generalized rash None None 1? 77
1 Occup., Cu–ammonia Bronchial asthma Cu/NH3 inhal.n Dyspnea 1 78
1 Dermatology patient Generalized uricaria Scratch, 1% aq. CuSO4 Erythema 2 2
(IUD)
1 Dermatology patient Pruritus and Scratch, CuSO4 Positive 2 79
(IUD)
urticaria
2 Patients (IUD) Rhinitis Cu prick test Positive 1–2 80
1 Patient (IUD; dental) Urticaria, flushing, Cu metal, 5% aq. CuSO4, Punctate wheals 1? 47
pruritus
actyl choline patch
1 Adhesive pads Urticaria, eczema Open/closed patch 5% Pos. to copper acetyl 3 76
aq. CuSO4
acetonate/urticaria; not to
Cu metal
1 Occup. exposure to Urticaria Copper metal 2% aq. CuSO4 Positive at 20 min. Positive 3 50
copper
at 24 hr
Abbreviations: CR, clinical relevance; IUD, intrauterine device.

128 Hosty´nek and Maibach
Table 3 Allergic Contact Dermatitis Due to Copper
Visible signs and
symptoms Patch test Challenge reaction CR References
Cases Cohort/etiology
3 Dermatol. patients Hand eczema Copper metal
Positive
0–1 43
Brass metal
Positive
1 Metal contact Dermatitis 1% aq. CuSO4 Positive patch 2–3 81
2 Dermatol. patients Dermatitis 10% aq. CuSO4 Positive patch 0–1 82
1 Teleph. lineman Dermatitis Copper metal Positive 1 44
1 Jewelry Eczema Copper coins
Positive at 72 hr
1–2 45
1.25–5% aq. CuSO4 Positive at 24 hr
1 Metalworker Eczema 2% aq. CuSO4, multiple Multiple positives, including 0–1 83
salts
Cu(II)
10 Furniture polishers Dermatitis 5% aq. CuSO4 Positive–irritation? 1? 84
1 Welder Eczema 0.1–2% CuSO4 pet. Positive at 72–96 hr 2 74
6 Dermatol. patients Eczema 0.25–5% CuSO4 pet. Positive to Cu and Ni 0 85
4 Agricultural Dermatitis 1% CuSO4, pet. Positive at 24–72 hr 0 19
2 Jewelry Dermatitis 5% CuSO4 aq. Positive to Cu and Ni; irr? 1? 86
140 Dermatol.
Dermatitis Copper metal Positive 0–1 49
patients/jewelry
1 Occupational Itching, eczema 2% CuSO4 aq. Necrosis at 24 hr 1 50
1 Dental patient Oral symptoms 1% CuSO4 pet. Positive patch 0–1 87
2 Dentistry students None 1% CuSO4 aq. Positive patch 1 88
1 Enameller Dermatitis 5% CuSO4, pet. Positive 0–1 89
3 Jewelry Dermatitis 2% CuSO4 pet. Positive to Cu and Ni 0–1 90
1 Painter Itching, edema 10% Cu pigment, in pet. Positive patch 1–2 91
5 Agricultural Dermatitis 2% CuSO4, pet. Positive at 48–96 hr 0–1 92
3 Dermatol. patients Oral symptoms 1% CuSO4 Positive patch 0–1 93
1 Dermatol. patients Dermatitis 2% CuSO4, pet.
Positive at 72 hr
2–3 53
Cu metal
Positive at 72 hr
Abbreviation: CR, clinical relevance.

Copper Hypersensitivity 129
Table 4 Systemic Allergic Contact Dermatitis Due to Copper
Visible signs and
symptoms Patch test Challenge reaction CR References
Cases Cohort/etiology
1 Dermatol. patient Lychen planus Cu metal, CuO 1% Positive at 96 hr, negative 2–3 33
(dental)
aq. CuSO4
1 Dermatol. patient Rash Cu metal Aerythema, vesicles, 0–1 46
(dental)
excoriation
1 Dermatol. patient (IUD) Generalized eczema 5% aq. CuSO4 Positive, negative to other 2 3
metals
1 Dermatol. patient (IUD) Generalized dermatitis 5% CuSO4 10% NiSO4 Positive 1 94
4 Dermatol. patients (IUD) Generalized dermatitis 2% aq. CuSO4 Positive patch 1 pos. to 2 4
NiSO4
3 Dermatol. patients (IUD) Generalized dermatitis, 0.01% CuSO4 Cu metal Pos. patch at 24/48 h. Pos. 1 48
itching
to Cu metal at 24 hr
1 Dermatol. patient (IUD) Generalized dermatitis 1% aq. CuSO4 Positive patch 2 95
1 Dermatol. patient (IUD) Pruritus and dermatitis 0.1%–1% CuSO4 Positive patch at 24 and 0–1 96
72 hr
3 Dermatol. patients (IUD) Eczema, edema CuSO4 None 1? 97
1 Dermatol. patient (IUD) Edema, erythema 1% aq. CuSO4 Vesicles and spongiosis 1 98
2 Dentistry students, staff, None 5% aq. CuSO4 Positive at 24 and 72 hr 0–1 99
patients
1 Dermatol. patient (IUD) Nephritis CuSO4 Pos. to CuSO4 and NiSO4 1 100
2 Dermatol. patients Oral lesions 5% aq. CuSO4 Positive patch at 72 hr 1 67
(dental)
1 Dermatol. patient Multiple metal
Cu and other metal Eczema 0–1 51
(dental)
sensitivities
patches
(Continued)

130 Hosty´nek and Maibach
Table 4 Systemic Allergic Contact Dermatitis Due to Copper (Continued )
Visible signs and
symptoms Patch test Challenge reaction CR References
Cases Cohort/etiology
1 Dermatol. patient Gen. oral eruptions 1% CuSO4 pet. Positive patch 0–1 101
(dental)
40 Dentistry students Metal allergies 1% aq. CuSO4 Positive patch 0 102,103
1 Dermatol. patient (IUD) Perimenstrual
0.01–1% aq. CuSO4 Positive patch, positive 1 52
angioedema
Cu metal
1 Dermatol. patient (IUD) Generalized skin Copper salt (?) Positive to copper and 0–1 104
reactions
silver salt
1 Dermatol. patient (IUD) Perimenstrual dermatitis 2% aq. CuSO4, other Positive 1? 105
metal salts
1 Dermatol. patient Lychen planus 2% aq. CuSO4, other Positive to Cu and other 0–1 68
(dental)
metal salts
salts at 4 days
3 Dermatol. patient Orodynia, lychen planus 2% aq. CuSO4 Positive at 48 hr 0–1 106
(dental)
Abbreviations: CR, clinical relevance; IUD, intrauterine device.

Copper Hypersensitivity 131
Recommended Screening Procedure in Suspected
Copper Urticaria
Open test: Application on healthy skin first and observation of the
test area for 60 minutes. If reaction is negative, on previously
affected skin (as suggested by patient’s anamnesis) spreading of
2% aq. CuSO4 on a 3 3 cm area. Immunologically mediated
reactions usually appear within 15–20 minutes, nonimmunologic
ones within 45–60 minutes after application (111). This difference
in delay is a major distinction between specific and nonspecific contact
urticaria. A positive reaction is seen as edema or erythema
(wheal and flare). A minimum of 10 na€ve background controls
with the test solution is suggested. A nonimmunologic reaction will
appear in the controls due to release of inflammatory mediators
from the cells without participation of specific IgE antibody (112).
A use test is suggested, handling the suspected agent and recreating
the original scenario inducing the reaction (108).
When open application is negative, a prick test with 2% aq. CuSO4
is suggested. A group of more than 10 background controls is
required in prick testing using physiologic saline solution to ascertain
that copper does not produce such lesions in normal controls.
The occluded application of a copper disk over 48 hours can also
confirm suspected sensitization.
In case of a positive test the open application may be repeated for
verification.
Confounding Factors in Copper Allergy Test Results: Cross-Reactivity,
Contaminants, Irritation, and Angry Back Syndrome
In many cases where copper allergy is suspected, positive patch tests to
copper (as metal or the sulfate) are equivocal, and assignment of clinical
relevance can be difficult or impossible because case reports in the literature
most often lack relevant details. One element of uncertainty in the diagnosis
of copper allergy is its cross-reactivity with other (adjacent) transition metals
in the periodic system of elements. Observations of multiple sensitivity to
metals have been made frequently, attributed to cutaneous or systemic contact
with alloys, and it is challenging for the investigator to ascribe the clinical
observation either to concomitant sensitization or to cross-reactivity. Often
patients react to compounds that are not the primary sensitizer. Originally,
Epstein (82) had raised the question of nickel and copper cross-sensitization
in 1955, and since then many cases of simultaneous sensitivity to nickel and
copper in the same organism have been reported (Tables 2–5). The immunologic
mechanism involved in hypersensitivity to multiple metals and crossreactivity
between copper and other transition elements has been investigated
in two independent in vitro studies and the event is well characterized now,

132 Hosty´nek and Maibach
Table 5 Population Studies of Copper Hypersensitivity
Visible signs and
symptoms Patch test Challenge reaction CR References
Cases Cohort/etiology
10 10 Furniture polishers Dermatitis 5% aq. CuSO4 Positive; irritation? 1? 84
0 37 Patients Dermatitis 5% pet. CuSO4 Negative – 113
0 1190 Dermatol. patients Eczema 2%–0.125% pet. CuSO4 Negative – 24
4 652 Agricultural workers Dermatitis 1% CuSO4, pet. Positive at 24–72 hr 0 19
140 964 Dermatol.
Dermatitis Copper metal Positive 0–1 49
patients/jewelry
1 10,936 Dermatol. patients Allergy 2%–0.01% aq./5% pet. Positive 1–2 114
CuSO4
1 190 Enamellers and decorators Dermatitis 5% CuSO4, pet. Positive 0–1 89
2 12 Dentistry students None 1% CuSO4 aq. Positive patch 1 88
32 60 Dentistry students Metal allergies 5% aq. CuSO4 Positive patch 0 102,103
5 46 Agricultural workers Dermatitis 2% CuSO4, pet. Positive at 48–96 hr 0–1 92
5 311 Dental patients Allergies Metals (?) Metals (?) 0 115
7 233 Metal workers Metal allergies 1% aq. CuSO4 ? 0 116
3 520 Dermatol. patients Oral symptoms 1% CuSO4 Positive patch 0–1 93
1 2660 Dermatol. patients Dermatitis 2% CuSO4, pet. Positive at 72 hr 2–3 52
Cu metal
Positive at 72 hr
1 60 Dermatol. patients Dermatitis 1% aq. CuSO4
Positive patch 2 40
Copper alloy

Copper Hypersensitivity 133
making it possible to put the numerous case reports on copper-induced allergy
in better perspective. Specifically, nickel ion-specific T-cell clones appear to be
recognized both by copper and palladium ions, but not by others such as
cobalt. This reactivity is likely to be favored by their bivalency and proximity
to nickel in the periodic table of elements. Investigations showed that among a
large panel of nickel-specific T-cell clones four different types of reactivity can
occur: reactivity to nickel only, cross-reactivity between nickel and palladium,
cross-reactivity of nickel to copper, or to both palladium and copper ion,
which both neighbor nickel in the periodic table of elements (117,118). In light
of these results, copper-positive patients are now more often screened for
allergy to other metals also, but only few among them are found to be truly
copper-sensitive. To illustrate the possibility of cross-reactions due to dental
materials in particular, a number of commercial alloys are tabulated, with
special attention given to the listed presence of copper and nickel (Table 1).
Purity of test materials can be a source of diagnostic equivocation with
the potential for false-positive results. Copper patch test material may contain
nickel as an impurity, as analytical grade copper sulfate was shown to
contain up to 0.002% nickel; high-purity copper wire in IUDs, which is also
used for skin testing, contain 0.0003% (3 ppm) nickel (24). Note that with
metal ACD in humans, highly sensitized subjects can react down to a few
parts per million of the hapten (119).
A potential cause of false-positive, clinically nonrelevant reactions that
can result in patch testing is hyper-reactive skin, also known as the excited skin
syndrome or ‘‘angry back’’ (120–122). This condition can result from multiple
inflammatory skin conditions or from strong positive patch-test reactions,
magnifying adjacent patch test responses or inducing nonspecific reactions. This
is a potential occurrence in testing for copper when several different metal
patches are simultaneously applied on the patient. Multiple positive reactions
may require separate, sequential tests with the involved substances.
Finally, several studies, especially those involving retrospective reviews
or large population groups, routinely examine skin reactions at 48 hours,
missing potential late-phase (72 hr) reactions after patch application (Table 5)
(33–39). They may result in false-negative diagnoses and under-reporting of
hypersensitivity to copper.
Determining Clinical Relevance
The open literature has been critically reviewed for clinical relevance of the
cases reported. A problem encountered often in the evaluation of diagnostic
tests from patients reacting to chemical substances is understanding the clinical
relevance of test results, because little or no data are reported to qualify
positive results. This becomes particularly difficult in the interpretation of
tests, which appear to indicate a compound as primary sensitizer that
is known to have no or little sensitization potential, such as copper.

134 Hosty´nek and Maibach
Benezra et al. have addressed the problem of classification by suggesting a
systematic analysis of available data to arrive at an expression of degree
of confidence in the results reported by investigators, thus to better define
morbidity of a putative allergen. A degree of confidence has been assigned
to all cases listed in Tables 2–5, listing the literature reports. Although
Benezra et al. (123) designed the system with skin contact sensitizers in
mind, which lead to delayed-type reactions, the approach appears more
generally valid and is applied to all cases reviewed here.
Criteria for Assignment of Degree of Confidence
Presence of vehicle-treated or untreated controls
Concentration of test substance judged sufficient to elicit a
response
Use of an appropriate vehicle
Purity of test reagent to exclude possible reaction to contaminants
Sufficient number of cases for meaningful response
The evidence provided in the reports is evaluated towards classification
of the agent (copper) as allergen and a degree of confidence on a scale
from 0 to 5 is assigned to indicate how well the test results demonstrate that
the chemical does or does not induce the immune reaction:
5 ¼ results meet all of the criteria
4 ¼ all criteria met, but number of cases is marginal
3 ¼ parameters such as controls are missing but reports point to
substance as sensitizer
2 ¼ controls are absent and there are no other details indicating
substance as sensitizer
1 ¼ results not considered to be reliable
0 ¼ test fails all of the criteria
Since evaluation of criteria is subjective, degree of confidence should be
viewed within a range of 1 of the number assigned. Listed in the following
are two categories of reports relative to copper hypersensitivity: populationbased
studies (also listed in Table 5), selected from published reports of immune
reactions to copper, which surveyed larger samples—random cross-sections of
the population, cohorts of specific occupational exposure, wearers of IUDs, dermatological
clinic data bases, or groups exposed to copper in dental materials.
That section is followed by selected case reports of more anecdotal value.
SUMMARIES OF POPULATION-BASED STUDIES
Barranco, 1972
Upon review of the literature the author noted six cases of ACD to copper:
three cases attributed to contact with brass, and one each to exposure to

Copper Hypersensitivity 135
copper sulfate, copper metal, and jewelry. The author also reported on a case
of dermatitis attributed to the use of a copper IUD. Although of questionable
clinical relevance due to patch testing with 5% CuSO4, it holds a
somewhat historical interest as it is the first report of eczematous dermatitis
to copper due to systemic exposure. Tested for the other frequent metal
allergens: Ni, Cr, Co, and Hg besides Cu, all patch tests were negative except
for a strong reaction to 5% CuSO 4. Remission was noted after removal of
the IUD (3).
Dhir, 1977
A cohort of 10 furniture polishers who had developed skin reactions on
handling ethyl alcohol tinted with 5% copper sulfate were tested with that
solution and aq. 5% CuSO4. All 10 patients reacted to both materials; the
test with the same materials were negative on 15 control subjects (84).
Jouppila, 1979
Assessed were 37 patients wearing copper IUD and presenting with skin
rashes. Epicutaneous tests for copper, nickel, and cobalt allergy showed
reactions to nickel (four) and cobalt (one), but none to copper. The authors
concluded that allergy to copper was not likely to be the cause of the side
effects (124).
Karlberg, 1983
Of 1190 eczema patients tested with serial dilutions (2–0.125%) CuSO4 in
pet. over a three-year period, none had a reaction to copper only, 13 reacted
to copper and other metals. Thus, no sensitization to copper specifically
became evident, leading to the assumption that the (multiple) reactions
noted were due to metals contaminating the test allergen. According to
Karlberg, highest-grade copper metal contains 0.0003% nickel, analytical
grade copper sulfate up to 0.002%. In the GPMT using dilution series of
0.1–0.01% CuSO4 for induction and 1–0.05% in pet. for elicitation, Karlberg
determined that copper sulfate was a grade I allergen. In her review of the
literature prior to 1982, Karlberg noted four relevant and 20 probably
relevant cases of copper hypersensitivity. Over 90 cases were classified as
uncertain or not relevant (24).
Lisi, 1987
The authors studied the prevalence of irritant or ACD from pesticides by
patch tests on 652 outpatients with skin disorders. Of 564 subjects tested
with 1% CuSO4, four cases showed positive reactions, none of which were

136 Hosty´nek and Maibach
irritant morphology. They presumed that allergic reactions could not be
considered of definite relevance due to the scarcity of clinical details. In particular,
data are missing on confirmatory retesting of positive tests conducted
2–3 months later (19).
Romaguera, 1988
Nine hundred and sixty-four dermatology patients complaining of metal
intolerance, experienced mostly in contact with jewelry, were patch tested
with standard allergens and metal washers. Of 52% of patients giving positive
reactions to nickel, 14% were also positive to copper (among other
metals), none to CuSO4 (concentration not given). The relevance of copper
sensitivity is uncertain due to the minimal experimental details given, and
the probability of contamination of the metal washers used (48).
Zabel, 1990
Records on 10,936 patch test reactions collected in a dermatology clinic over
the period 1975–1985 were reviewed, in addition to patch tests conducted on
118 patients wearing IUDs. Besides the record of patients with positive reactions
to multiple metals (mostly nickel), one eczematous IUD-wearing
patient reacted to CuSO4 at 5% in pet. only. After removal of the IUD
the eczema resolved. The causative role of copper is uncertain due to lack
in supporting evidence in that case (116).
Motolese, 1993
The authors reported on skin sensitization to metals encountered in a cohort
of enamellers and decorators. Relevance of the only positive reaction to copper
was uncertain due to the high concentration of 5% CuSO4 used in the
test. Also, too few clinical details were given to establish a firm causeand-effect
relationship in that case (89).
Kawahara, 1993
The cause of occupational allergies was investigated in a dental technology
school by testing a cohort of 12 students with 40 potential contact allergens
occurring in the manufacture of prostheses and was determined to be dust,
mist, and fumes in their environment. Two reacted to 1% aq. CuSO4; the
reactions could not be assessed as to their clinical relevance due to lack of
any further details (88).
Tschernitschek, 1998
Over the period 1982–1997 in a dental clinic, of 311 patients who were patch
tested for dental materials-induced hypersensitivity, 13% showed positive

Copper Hypersensitivity 137
reactions. Most frequent among the sensitizing materials were metals (77 of
107). Three among those reacted to copper and cadmium, two to copper
only. Significance cannot be assigned due to total lack of experimental
details (115).
Candura, 1999
Of 233 ACD outpatients patch tested with the standard GIRDCA test series
in a dermatology clinic, three had positive reactions to copper along with
other metals, four to copper only. The importance of the causative role of
copper cannot be assessed due to a total lack of experimental details (116).
Vilaplana, 2000
A testing program including 520 patients with dental prostheses who presented
with adverse oral mucous membrane reactions was conducted using
a special metal test series that included 1% CuSO4 in pet. Of 289 patients
with one or more positive reactions, one patient only reacted to copper, classified
as a reaction of past relevance (sic) by the authors; two patients had
reactions to copper with unknown relevance (93).
Wöhrl, 2001
In the endeavor to assess the relevance and diagnostic value of positive reactions
to copper, 2660 routine patch tests recorded in an allergy clinic over
2.5 years were screened for positive reactions to copper (2% CuSO4 in pet.)
and the other metals in the immediate vicinity in the periodic system of
elements: nickel, palladium, cobalt, and mercury. Of 94 cases that were
copper-positive, 26 were enrolled in a retest program involving CuSO4 at
5%, 2%, 1%, 0.6%, 0.2%, and 0.05% aq. Testing with copper foil was also
included. Of the original 26 inductees, 10 were positive to copper on retesting
with 5% CuSO4 in pet., but eight of those also reacted to a nickel patch.
Two of 10 showed unequivocally positive reactions to 2% CuSO4 in pet. Two
were positive to copper foil. Only one case showed an isolated sensitivity to
copper and not to any of the other test allergens, presenting with chronic
eczema of the fingertips. That patient’s occupation as electrician would
characterize the case as ACD to copper induced through cutaneous contact.
One other patient with multiple metal sensitivities appeared to have clinically
relevant sensitivity to copper. Presenting with eczema to a golden ring
(test to gold negative), the condition resolved when the patient exchanged
the gold ring with one made of silver. Although authors concluded on
copper–nickel cross-reactivity on the T-cell level in 9 of the 10 cases, with
a high statistical association, and copper sensitivity being of low clinical
relevance, all reactions cleared at 96 hours, a delay that is typical for irritant
reactions rather than ACD (52).

138 Hosty´nek and Maibach
SUMMARY OF SELECTED CASE REPORTS OF IMMUNE
REACTIONS TO COPPER
The process of diagnosis and remediation of copper allergy recorded in the
literature so far follows an overly direct and simple path, with little or no
clinical data. Solidly worked-up cases are rare, which makes assignment
of relevance difficult and is the reason for the low scores in clinical relevance
assigned to the reported cases in Tables 2–5. In most cases the examining
physician (dermatologist), having confirmed ACD, often by CuSO4 patch
test only, without controls or tests with other metals, advises the patient
to remove amalgam or antifertility device. As a rule, then, improvement
of the condition or complete remission follows promptly. As the signs of
allergy are no longer present, no follow-up testing is done. Cases where
no improvement was noted have not been published. With few exceptions,
sensitization to copper results mostly from two types of exposure: leaching
of the metal from dental amalgams, and from copper containing IUDs, due
to corrosion in the physiological environment. The majority of signs and
symptoms of allergy reported point to the delayed type, ACD, or systemic
allergic contact dermatitis (SACD).
At first glance, the literature search produced eight records with nine
cases of ICU (Table 2), 21 records with 160 cases of ACD (Table 3), and
21 records with 71 cases of SACD (Table 4). Many of the cases, diagnosed
by using a 5% CuSO4 patch in pet., are classified as having borderline relevance,
because patch tests at that strength are now considered to cause
probable irritation—rather than ACD (3,89).
A few of the cases listed in the tables merit detailed discussion due
to their extraordinary characteristics. A number of them also show that
sensitization originally thought to be due to copper actually resulted from
exposure to other metal(s) or chemical agents.
SELECTION OF INDIVIDUAL REPORTS OF IMMUNE
REACTIONS TO COPPER
Frykholm, 1969
An example of SACD due to dental materials was reported by Frykholm et
al. A patient with oral lichen planus-1ike reactions to dental restorative
materials showed positive skin reactions to epicutaneous tests with copper
metal, Cu (II) hydroxide, Cu (II) oxide, and Cu (I) oxide at 72 hours, while
the test sites were unreactive at 48 hours. Tests carried out on 20 controls
were negative. The patient’s oral lesions flared up in conjunction with the
skin tests. On repeated retesting at month-long intervals with metallic copper
and Cu (II) oxide, positive reactions to all test materials were until the
fourth day (33).

Copper Hypersensitivity 139
The late appearance of the skin reactions (24–96 hr) is characteristic of a
‘‘delayed allergic reaction,’’ to gold in particular due to its anti-inflammatory
activity (125,126). The delays in tissue involvement seen with copper may also
be ascribed to that characteristic, which makes the metal an effective antirheumatic
agent. An alternative explanation for the delayed reaction may
involve the protein reactivity and depot formation by copper, resulting in
delays in SC penetration as seen in other electrophilic metals, e.g., aluminum,
silver, mercury, or chromium known for their retention in the SC (127–131).
Shelley, 1983
A case of potential contact urticaria, which defies interpretation, was
reported for a woman with occult sensitivity to copper. Wearing an IUD
and dental fillings with copper amalgam, she developed urticaria and flushing
with pruritus upon physical exercise, emotional stress, or overheating,
more severe during the perimenstrual period. On testing, metallic copper
and copper-containing coins elicited no response, but on the test sites she
developed punctate wheals when challenged with exercise or on injection
with acetylcholine in subthreshold concentration. Also challenge with 5% aq.
CuSO4 induced the same response as the copper metal. Tests with nickel
metal and the sulfate were negative. That cholinergic urticaria reaction
observed is attributed to a collaborative effect of acetylcholine and copper
on mast cell membranes inducing degranulation, a clinical response that an
antibody–antigen reaction on the mast cell surface alone could not produce.
No subsequent such patients have been reported (46).
Hocher, 1992
A woman with IUD presented with interstitial nephritis and renal failure.
Patch tests were positive for copper, nickel, and cobalt. An in vitro
lymphocyte-stimulating test with copper was also positive. On removal of
the contraceptive device renal function returned to normal. No similar cases
have been published (100).
Laubstein, 1990
A case of occupational exposure to copper metal presented with an itching
eczematous condition, which healed on absence and returned on resumption
of work. That highly sensitized individual reacted with necrosis on 24-hour
testing with 2% aq. CuSO4, and also on contact with copper metal. Followup
brought an urticarial reaction after 20-minute exposure to copper metal,
and to serial dilution of aq. CuSO4 after 24 hours, reactions that extended
to necrotic ulceration. Upon changing occupation the patient became free
of symptoms. This description suggests two mechanisms—ACD documented
with a positive response to a copper disk, and immunologic contact
urticaria (49).

140 Hosty´nek and Maibach
Sterry, 1985
A female patient reported with eczema and pruritus while wearing selfadhesive
pads treated with the disinfecting agent copper-acetyl acetonate.
Reactions to patches with copper-acetyl acetonate on 12 control subjects were
weakly positive at 24 hours, disappearing after 48 hours. The test with metallic
copper and CuSO4 on the patient was negative, but positive to the disinfectant
at 50% pet. and 50% aq. On open patch testing, the patient was positive to
both copper-acetyl acetonate 50% aq. and acetyl acetone (100%) alone. This
suggests that allergy was probably due to acetyl acetonate and not copper (76).
COMMENTS
Many case reports of sensitization attributed to copper may be difficult to
classify as such with certainty. Copper sensitivity may overlap with nickel
hypersensitivity, or nickel alone may even be the only causative agent, as
in dermatological or dental practice they can only be distinguished with
difficulty when assessing exposure in the individual patient. As results from
several in-depth investigations, patients with a positive test to copper also
appear sensitized to nickel, and vice versa. This can be attributed to cellbiological
and metallurgical factors:
Investigations at the cellular level have established cross-reactivity
between the two metals, which may account for the frequency of
copper hypersensitivity reported (117,118).
At the exposure level, often copper and nickel are associated in IUDs
or orthodontic materials. Copper of highest purity still contains traces of
nickel; thus, sensitization observed may be concomitant (24,124,132).
In dermatological practice, diagnostic test materials copper sulfate
or copper metal discs also contain low levels of nickel sufficient to
elicit a reaction in an organism highly sensitive to nickel, leading
to a false-positive diagnosis.
There may be true allergic reactions to copper exposure, topical or systemic:
to copper salts, to the metal, or to its alloys. Judging from the cases
reviewed so far, such responses are rare.
CONCLUSIONS
Systemic as well as topical exposure to copper can cause both immediate
and delayed-type sensitization. Contact dermatitis and urticaria attributed
to copper metal or its compounds has been suggested, with effects from
dental materials and IUDs as the main etiological factors. Immune reactions
occurring in industry are few, considering the number of copper smelters

Copper Hypersensitivity 141
and refinery workers in daily contact with the metal. The majority of sensitization
reports may be due to copper cross-reactivity with nickel and
palladium. Thus, true allergic reactions to copper appear rare, particularly
those induced by skin contact, which is consistent with copper’s rating as
a grade I allergen in the guinea pig maximization test. Most cases of confirmed
copper allergy result from its presence in orthodontic materials,
and those reactions are mostly of the delayed type.
Firmer chemical and epidemiologic judgments will be possible when:
1. Additional experimental data become available on the nonirritating
dose(s) suitable for diagnostic patch testing (in petrolatum and
water), and in water for prick testing. On the basis of Wöhrl’s
data, 2% in petrolatum may be appropriate (52).
2. Authors describe their clinical experimental data with details of
the several steps as documented in the Operational Definition
of ACD (56), specifically re-patch testing upon indication, serial
dilution patch testing, and use testing (PUT/ROAT). Those steps
will help clarify clinical relevance.
ABBREVIATIONS
ACD allergic contact dermatitis
aq aqua
ICDRG International Contact Dermatitis Research Group
ICU immunologic contact urticaria
IUD intrauterine device
LLNA local lymph node assay
pet. petrolatum
PUT provocative use test
ROAT repeat open application test
GPMT guinea pig maximization test
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81. Gaul LE. Incidence of sensitivity to chromium, nickel, gold, silver, and copper
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146 Hosty´nek and Maibach
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Copper Hypersensitivity 147
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148 Hosty´nek and Maibach
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8
Copper in Medicine and Personal
Care: A Historical Overview
Roberto Milanino
Facoltà di Medicina e Chirurgia, Sezione di Farmacologia,
Dipartimento di Medicina e Salute Pubblica, Università di Verona,
Verona, Italy
Remember always that some ideas that seem dead and buried may at
one time or another rise up to life again, more vital than ever before.
—Louis Pasteur
INTRODUCTION
In the pre- and protohistoric ages, medicine and therapy were both strongly
linked with religion and thus were mainly presented to communities and
patients in magic-religious clothing, although empiricism actually lay at
the core of these arts.
Medicinal plants were widely employed as drugs, and, in some cases,
their active principles are really valuable and still used clinically today, e.g.,
atropine (Atropa belladonna, Datura stramonium—L.), digitalin (Digitalis
purpurea—L.), opium and derivatives (Papever somniferum—L.), quinine
(Chincona offinicinalis—L.), reserpine (Rauwolfia serpentina—L.), salicin
and derivatives (Salix alba—L.), tubocurarine (Chondrodendon tomentosum—
R. & P.), etc. Drugs from the animal kingdom were numerous, such as the
donkey fat, rhinoceros horns, and pig liver; the latter, for instance, was
149

150 Milanino
considered a good remedy for anemia (an empiric indication consistent with
our modern medical knowledge). Minerals, for example those containing
antimony, arsenic, copper, gold, iron, lead, mercury, sulfur, or zinc, were
also used frequently. Many of the above ingredients were routinely mixed
according to magic-empirical criteria in recipes, often kept secret, and usually
administered accompanied by complex esoteric rituals.
The aim of this review, however, is focused on the use of copper in therapy
during the prescientific millennia preceding our modern medical culture.
Humans have employed copper in medicine since before 3500 B.C., mostly
referring to it as a ‘‘generic drug,’’ but also recognizing its antiseptic as well
as anti-inflammatory potentials. In particular, attributing anti-inflammatory
properties to copper appears to have been an extremely clever intuition. What
is even more surprising is that this metallo-element was believed to be endowed
with therapeutic properties by almost all major ancient cultures. In particular,
while its diffusion among Mediterranean, near-, middle-, and far-Eastern civilizations
may be explained on the basis of their frequent mutual contacts, the
presence of copper in the ‘‘pharmacopoeia’’ of the pre-Columbian Meso- and
South-American cultures seems to derive entirely from the autochthonous
experience of these populations.
THE SUMERIC CULTURE: CIRCA 4000–2300 B.C.
Present day knowledge ascribes to this culture the basic merit of having invented
the near- and middle-Eastern ideographic writing, which is conventionally considered
as the beginning of the historical age. This society developed recurrent
commercial and cultural relations with the neighboring areas and cultures (1).
Malachite (basic cupric carbonate) was one of the products that
Sumerians frequently exported to Egypt as well as closer regions, mainly
for making jewels and cosmetic products, and also for pharmaceutical preparations.
It is documented, through indirect evidence (mostly Egyptians),
that the utilization of pulverized malachite for generic medical purposes
was a practice commonly used by the Sumerians themselves (2).
THE ANCIENT EGYPTIAN CULTURE
The Predynastic Age to the II Intermediate Period (XVII Dynasty):
Circa 3900–1550 B.C. (3,4)
Probably the first significant medical document known today is the Kahun
papyrus, which was most likely written during the kingdom of Seosostris II
(about 1880 B.C., XII dynasty), and is essentially focused on gynecological
problems (4,5). The Kahun papyrus appears to make no specific mention of
the use of copper as a drug.
However, we know from other minor written fragments of those times,
as well as numerous and much more exhaustive subsequent medical

Copper in Medicine and Personal Care 151
manuscripts, that the utilization of pulverized malachite was extremely common,
since the beginning of the predynastic period, for preparing a typical
blue-green eye makeup (5).
In the middle and late period of this Egyptian age, this cosmetic
became dark gray, as shown by several tomb pictures, probably because
pulverized galena (lead sulfide) was already added to malachite (2,6). This
eye makeup was used not only for decorative ritual purposes exclusively
related to the Osiris cult but also for the prevention and cure of eye infections,
widespread in the ancient as well as modern Egypt. These infections
were mainly caused by the dryness of the climate and sandy winds characterizing
the area (2,5–7). Actually, in this practice, it may be possible to
recognize the first empiric attribution of antiseptic properties to copper
preparations.
The XVIII Dynasty to the Ptolemaic Dynasty: Circa
1550 B.C. to 30 A.D.
The most famous and comprehensive documentation of Egyptian medical
science are the Ebers’ and Smith’s papyri. They summarize medical information
of those times based on, if not mainly, previous orally transmitted and
written traditions. Both these treatises belong to the XVIII dynasty, the first
of the New Kingdom (1550–1075 B.C.) (5,7).
The papyrus of Ebers (circa 1500 B.C.) is a true medical encyclopedia,
dealing with both generic medical and therapeutic problems. The pharmacological
sections of the Ebers’ papyrus emphasize the importance of
personal cleanliness, and reports the use of many drugs mainly coming from
medicinal plants (castor oil, senna, tamarisk, thyme, etc.), but minerals are
also not neglected. For example, this text clearly codifies, for the first time,
the preparation of kohl, i.e., the mixture of malachite and galena. This compound
is certainly identical to that used in previous centuries, and is still
routinely applied as an eye cosmetic, for the same decorative–ritual and
practical medical purposes as described earlier (2,5,6).
The papyrus of Smith (about 1450 B.C.) mainly addresses surgical problems
as Egyptian society (like the later Roman one) was frequently involved
in wars and, consequently, was forced to develop efficient emergency
medical procedures (5). This papyrus also mentions the use of pulverized
malachite thought to be endowed with astringent, healing, and antiseptic
properties for the treatment of postoperative wounds (2,5). Interestingly,
these antiseptic and healing potentials of copper are presently fully recognized
(8), although no longer routinely employed in modern medical practice.
However, copper preparations, in particular cupric sulfate and basic cupric
sulfate (9), are still widely used in agriculture; copper is also a potent antimycotic
agent, especially effective in the treatments of plants, such as
tomatoes and grapes, against fungal infections.

152 Milanino
THE BABYLONIAN–ASSYRIAN CULTURE: CIRCA 1750–539 B.C.
The medical and pharmacological knowledge of these societies is reported
on approximately 800 medical clay tablets recovered from circa 100,000
found during the excavation of the Assyrian Library of Nineveh
(about 650 B.C.) (2,10). Babylonian–Assyrian medicine particularly focused
on studying the liver as a basic tool of its diagnostic. This organ, however,
was not examined according to modern semeiotic procedures (directly on
patient), but instead physicians examined the livers as well as the viscera of
animals sacrificed during the propitiatory rituals performed in order to recognize
the disease, then chose the appropriate therapy (2). This practice was
clearly an esoteric superstition called Haruspecism. Most likely, Haruspecism
reached its highest levels over 900 years later with the development of the
Etrurian culture in Italy (11). In fact, the Etrurian haruspices not only ‘‘specialized’’
in this kind of ‘‘medical approach’’ but also transformed it into a more
general divinatory procedure, which included the magically inspired interpretation
of many other natural phenomena, such as the flight of birds (11).
About 250 drugs of plant origin, and also 120 compounds derived from
minerals, are cited in the Babylonian–Assyrian pharmacopoeia. Among the
minerals, those containing copper are mentioned specifically, being qualified
as generic therapeutic remedies (2). However, most likely for the first time,
the use of a copper bracelet is quoted in these reports as useful, albeit nonspecific,
pharmacological tool (10).
THE ANCIENT INDIAN CULTURE: CIRCA 2800–1000 B.C.
The medical knowledge acquired since the remote origins of the ancient
Indian culture and orally transmitted generation to generation was later
summarized in two major medical books: the Samhita Charaka and the
Susruta Samhita (written in Sanskrit during the Brahamanic age, approximately
between 800 B.C. and 1000 A.D.) (1,2). Surgery was highly developed
in the ancient Indian culture and, unsurprisingly, their pharmacopoeia made
a wide use of medicinal plants, among which is mentioned Cannabis indica
L. as an analgesic (cannabinoid derivatives). This drug was frequently
employed, together with black henbane (Hyoscyamus niger—L.), which contains
such analgesic–narcotic principles as hyoscyamine and scopolamine, as
a general anesthetic in surgical operations (2).
Although no other significant information is available, the use of
copper (as sulfide or sulfate) for making preparations used for nonspecific
medical purposes is reported in these texts (2).
THE ANCIENT CHINESE CULTURE: CIRCA 3000 B.C. TO 1100 A.D.
According to legend, the medical book Nei Ching (‘‘The Canon of Medicine’’)
was written by the mythical king Huang Ti (the ‘‘Yellow Emperor’’) about
2700 years B.C. (2). Actually, this treatise was most likely edited much later

Copper in Medicine and Personal Care 153
and, moreover, appeared in two separate parts, about 10 centuries apart.
In fact, the first part dates back to approximately the second century B.C., the
second part to about the eighth century A.D. However, ‘‘The Canon of
Medicine’’ is the first-known compendium in which millennia of Chinese
medical and therapeutic traditions are summarized (2).
The pharmacological use of copper sulfate (or sulfide) is undeniably
documented in the above text, not only for the topical treatment of skin
and eye diseases but also for ‘‘blood purification.’’ The latter is, probably,
the first-known indication for oral administration of copper (2).
THE PRE-COLUMBIAN MESO- AND SOUTH-AMERICAN
CULTURES: CIRCA 600 B.C. TO 1500 A.D.
The medical traditions of the better-known Meso- and South-American civilizations
(i.e., Maya, Aztec, and Inca) are referred to in numerous documents,
albeit those written exclusively by the Spanish invaders (2).
In particular, the cranial trepanation procedure deserves an especial
attention. Although known and occasionally practiced since at least the fifth
to fourth millennium B.C. among most of the world’s prehistoric and historic
cultures, including that of Egypt, this kind of surgery was chiefly practiced
by Meso- and South-American societies between the fifth century B.C. and
the fifth century A.D. (12,13). Craniotomy, however, reached an astonishing
level of specialization among the Inca ‘‘surgeons,’’ who frequently made use
of this peculiar technique and whose patients actually had an extraordinary
survival rate, assessed far beyond 50%. In fact, archeologists discovered
skeletons of a great number of Incas whose skulls underwent this procedure,
and who survived the distressing experience (12). Interestingly, a human
cranium found in the necropolis of Cuzco (Peru) clearly shows the holes
resulting from two successive cranial trepanations; obvious signs of bone
regeneration surrounding these holes testify to the individual’s long survival
following the surgical procedures (12). The main purpose of this intervention
was undoubtedly a ritual–magical one (2,12,13); nonetheless, the
craniotomy also bears real clinical value in reducing intracranial pressure
due to post-traumatic meningeal edemas or hemorrhages (10).
Notably, ‘‘gauze’’ soaked in a copper sulfate solution was routinely used
to ‘‘disinfect’’ the surgical wounds after the removal of the skull section (2,13).
THE ANCIENT GREEK CULTURE
It is relevant to note that the chronological classification of Ancient Greek
society and culture is more conventional than factual. For instance, the
dating of Archaic Greek, of the classic Greek, and, especially, of the official
end of this unique civilization (which is made to coincide whit the Roman
conquest, i.e., 323 B.C.) is merely symbolic. In fact, as we will see below,

154 Milanino
the Greek culture developed and survived much longer, and is still largely
present in the roots of our contemporary occidental civilization.
Archaic Greek: Circa 1300–500 B.C.
Although the ancient Greeks attributed the beginnings of their medicine to
the probably mythical Thessalonian prince Asclepios (about 1200 B.C.), earlier
documented Greek medical records came down from the Mediterranean-
Greek colonies, especially the Sicilian one (11,14).
Actually, significant medical theories were derived from observations
of ancient Greek scholars. For instance, Alcmeon (circa 560 B.C.) postulated
that the brain was the center of human sensorial and intellectual life, and
Empedocles (an Alcmeon’s contemporary) speculated that respiration occurred
both at the pulmonary and skin pore levels (11).
It was probably in those times that the use of copper preparations,
most likely borrowed from the Mesopotamian, Egyptian, and Minoan medical
traditions, was introduced as a nonspecific remedy (11,14).
Classic Greek: Circa 500–323 B.C.
About 100 years after Alcmeon’s and Empedocles’ age, when classic Greek
culture developed under the influence of the thoughts of the great philosophers
(such as Socrates and Plato), Hippocrates (460 B.C.) was probably the
first physician to invoke the separation of medicine, religion, and myth, stating,
‘‘No disease is more divine or more human than any other, since every
illness is due to a natural cause, in the absence of which it cannot take place’’
(11). The Hippocratic perspective on human brain functions was also very
modern; redefining the previous hypothesis of Alcmeon, Hippocrates wrote,
‘‘I say that the brain is the most powerful organ of the human body ...The
eyes, ears, tongue, hands and feet all act under the control of the brain.
The brain transmits its messages to the human conscience’’ (11,14). Nevertheless,
this does not necessarily mean that Hippocrates’ attitude had been a sort
of ‘‘revolutionary’’ one. In fact, Hippocrates was especially careful not to
express open dispute with the contemporary religious opinions and traditions,
and in the prologue to his famous ‘‘Hippocrates’ swearing,’’ Hippocrates
wrote, ‘‘I swear in the name of Asclepios, the Physician, Igea, Panacea, and
all the gods and goddesses, calling them to witness, that I will follow ...’’ (11).
Hippocrates’ major work is the Corpus Hippocraticum, which consists
of 72 volumes, of which only 17 are sure to have been written by Hippocrates
himself. The other 55 are ‘‘contaminations of ’’ and ‘‘additions to’’
the original texts by numerous respectable physicians (but also by some
quack doctors) in the course of following centuries (11,14).
In Hippocrates’ original books, the use of copper preparations was
clearly prescribed for the therapy of many such diseases as cutaneous and
eye diseases, vaginal disorders (using an irrigation with copper solutions or suspensions),
and hemorrhoids (using ‘‘suppositories’’ containing copper) (11,14).

Copper in Medicine and Personal Care 155
Finally, ancient Greek medicine was the first to suggest the wearing of
the copper bracelet as an antiarthritic remedy, which brings us directly to
our present debate on the effectiveness of topical copper preparations in
the treatment of rheumatic conditions (14,15).
THE ANCIENT ROMAN CULTURE: CIRCA 600 B.C. TO 476 A.D.
From the beginning, ancient Romans were primarily engaged in bringing
their ‘‘Pax Romana’’ (the archetype of our contemporary globalization process)
to all societies living in the known world. Thus, the Roman age based
both the origin and development of its knowledge, the medical one included,
on Greek thought and tradition, although initially some Etrurian and later
Arab contributions were also present (11). Therefore, in spite of the much
older origin of Roman society and culture, the major works describing
Roman medicine and pharmacology were all written between the end of
the first century B.C. and the second century A.D., and are attributable to
three prominent names, i.e., Pliny ‘‘the elder,’’ Celsus, and Galen (11).
Caius Pliny ‘‘the elder’’ (23–79 A.D.) wrote the 37 volumes of Naturalis
Historia, in which Pliny discourses not only upon the subjects of astronomy,
geography, zoology, and botany, but also on medicine, physiology, and
pharmacology. Pliny’s pharmacopoeia, along with medicinal plant and
animal derivatives, devotes significant attention to minerals, copper in particular.
This metal is frequently mentioned in many preparations in which it is
present in the form of pharmacologically active compounds such as copper
oxides, copper sulfide, copper sulfate, basic cupric carbonate, and basic cupric
acetate (C.P.E., Nat. Hist. XXX and XXXI) (16). Their practical use was
particularly recommended for the treatment of eye and skin diseases, tonsillitis,
throat inflammation, etc. (C.P.E., Nat. Hist. XXX and XXXI) (16).
Aulus Cornelius Celsus (probably, 30 B.C. to 38 A.D.) was the author of
De Medicina, an eight-volume encyclopedia, in the third tome of which,
treating on ‘‘the fevers,’’ Celsus codified his famous ‘‘four cardinal signs
of inflammation,’’ rubor, tumor, calor et dolor, to which a fifth sign, the fuctio
laesa, was then added by Galeno (17,18).
It was again Celsus who, probably first, formally recognized copper as
useful antiseptic and, especially, anti-inflammatory agent; notably, the antiinflammatory
potential of basic cupric carbonate (i.e., malachite) has been
confirmed by our contemporary research (19). Copper compounds were
indeed frequently employed in Celsus’ very elaborate prescriptions such as
those suggested for the treatment of anal rhagades, hemorrhoids, tonsillitis,
wound disinfection, etc. We like to report as an example, Celsus’ recipe for
curing anal rhagades:
Verdigris (basic cupric acetate): parts 2
Myrrh: parts 12
Antimony: parts 16

156 Milanino
Poppy ‘‘tears’’: parts 16
Acacia: parts 16
To be suspended in wine before local application (17)
In the writings of Celsus, a great deal of attention is also dedicated
to surgery. In fact, Celsus first introduced in orthopedics the application
of copper plates (which was thought to be appropriate for those conditions,
since they are also antiseptic and anti-inflammatory) to promote the recovery
of disassembled fractures (17). Interestingly, this practice was still in use
more than 1400 years later, as exemplified by a human skeleton recently
discovered in Belgium (cathedral of Vrasene) (17).
Claudius Galeno (129–200 A.D.) wrote many medical books such as the
Ars magna and the Ars parva, as well as works of pharmacology collected in
the treatises De Methodus Medendi and De simplicium medicamentorum
temperamentis et facultatibus (11). Galeno, a Civis romanus (although fully
Greek by origin, culture, and thought), was an eminent and innovative
anatomist, physiologist, and pathologist, but a less pioneering therapist
(18). In fact, while Galeno’s ‘‘general medicine’’ writings describe some sort
of interesting experimental work, Galeno’s pharmacology books are mainly
a summary of therapeutic experiences gathered by the ancient Mediterranean
and Eastern cultures (the Greek one in particular), skillfully mixed
with the pharmacological reports of Celsus as well as Pliny ‘‘the elder’’ (18).
Thus, copper was also frequently used in Galeno’s therapeutics, but
still on the basis of an empirical methodology, using essentially the same
copper preparations, and being addressed to the same pharmacological targets
as in the previous Mesopotamian, Egyptian, Chinese, Indian, Greek,
and, of course, Pliny’s and Celsus’ heritages. Galeno’s pharmacopoeia is,
therefore, devoid of any original ‘‘scientific’’ approach, perhaps with one
exception: occasionally, Galeno’s ‘‘new’’ drug mixtures were tried to test
in self-experimentation (11,17,18).
Nevertheless, Galeno’s works were destined to play a significant role in
the future trends of medical and pharmacological scientific ‘‘development’’
over the following 1500 years, especially in our European ‘‘world,’’ and also
in the Arabian one (18).
FROM THE HIGH-MEDIEVAL AGE TO THE EARLY
20TH CENTURY
The classic Greek and Roman ‘‘biomedical’’ background deeply affected
biological, medical, and pharmacological thought at least up until the
‘‘Galilean revolution,’’ which generated a totally new way of seeing and
doing science (second half of 16th, first half of 17th centuries) (11,18,20).
Thus, different fragments of Hippocrates’, Pliny’s, Celsus’, and Galeno’s
writings were blended according to current ideas and prejudices, mainly in

Copper in Medicine and Personal Care 157
consequence of the dogmatic and actually prevailing Catholic culture, which
found its major justification in Scholastic philosophy (a re-reading of
Aristotelian thought), as elaborated by Thomas Aquinas (13th century A.D.)
(20,21). Notably, in this discouraging context, remarkable attention was dedicated
to Galeno’s works, since Galeno possibly believed, and certainly stated,
‘‘the human body is merely an instrument of man’s soul’’ (11,18,20).
Nevertheless, the pharmacological use of copper survived during those
dark centuries, although essentially based more on the imaginative superstitions
derived by the ‘‘innovative discoveries’’ of contemporary quack
doctors and alchemists, than on the ‘‘good old’’ empiricism that characterized
the progress in medicine and pharmacology made by scholars of the
ancient cultures, particularly classic Greek–Roman.
Later on, the ‘‘Galilean revolution’’ also began to strongly influence
the world of biology and medicine and the related biomedical disciplines
slowly evolved into our modern experimental development process of scientific
learning. Pharmacology, and especially copper, however, were destined
to wait still a while longer to see their ‘‘renaissance.’’
In fact, focusing on copper, it was only in the first half of the 19th century
that Rademacher, introducing the so-called ‘‘medicine of experience,’’
deduced, after a clinical trial lasting 25 years, ‘‘the body has a strange predisposition
for a great number of diseases which disappeared, or could be
healed, in the presence of copper. Healthy subject did not show any response
to copper’’ (18,22). At the end of the 19th century, copper was administered
internally and was found to be effective against numerous illnesses such as
skin diseases and the infections of tuberculosis and syphilis (22). The roots
of these clinical uses of copper, however, were again to be found in a sort of
‘‘renewed empiricism,’’ which, according to Classic Age traditions, considered
copper a valuable antiseptic and anti-inflammatory agent.
Between the end of the 19th and the beginning of the 20th centuries,
mounting consideration for the discoveries of modern microbiology, physiology,
and biochemistry, as well as the appearance of chemistry as science
in the field of pharmacology (e.g., the synthesis of arsenic derivatives, salicylates,
sulfanilamides, etc.) (18,22), led to the abandonment of the use of
copper in medicine. It is true that during the 1940s some copper complexes,
such as Cupralene and Dicuprene, were still employed for treating arthritic
diseases, but the advent of the therapy with exogenous cortisone extracts or
derivatives rapidly led also to their disuse.
BEGINNING OF THE SCIENTIFIC AGE FOR COPPER: 1928–1976
Curiously, it was only in the first half of the past century, while copper therapy
was gradually being neglected by a majority of clinicians, that the
importance of copper in biology and medicine finally found its experimental
justification.

158 Milanino
In fact, in 1928 copper was first shown to be essential for life (23) since
it is required for the synthesis of hemoglobin. Subsequently, its involvement
in inflammation was demonstrated both in humans (1938) and laboratory
animals (1953) (24,25). Almost 30 years after the scientific reports above,
in 1967, a dramatic increase of the concentration in ceruloplasmin (the
major multifunctional copper protein present in the serum, synthesized by
the liver and deeply involved in iron metabolism and copper delivery to the
extrahepatic tissues) was observed in the serum and periodontal tissue
specimens of patients suffering from periodontal disease (an acute inflammatory
process) (26,27). In 1968, a relevant increase of serum copper and
ceruloplasmin was also reported in rheumatoid arthritic patients (28). Subsequently,
the same evidence was found to characterize the adjuvant arthritis
of the rat, an experimental systemic disease still the best existing model
of human rheumatoid arthritis (29,30).
All the above were only occasional reports, however. The leading
breakthroughs that characterized the beginning of scientific development
of ‘‘copper and inflammation’’ research came in 1976, when Sorenson’s (31)
classic paper indicated many copper complexes as active acute and chronic
anti-inflammatory agents, and when Whitehouse (32) posed the question
about a possible ‘‘ambivalent,’’ i.e., pro- and anti-inflammatory, role of
copper in the development and control of inflammation.
CONCLUSIONS
Today, the fact that ‘‘endogenous’’ copper exerts a key role in the development
and control of inflammation is a well-recognized biological
phenomenon (33). Moreover, the anti-inflammatory/antiarthritic potential
of ‘‘exogenous’’ copper in curing experimental models of inflammation has
also been demonstrated repeatedly by many research groups (34,35). Recently,
great progress has been made, at the molecular level, in understanding
how copper ‘‘traffics’’ in the organism and within the cells. Although this
basic topic of research is still in its infancy, in the near future it may yield
essential information for the understanding of the mechanism(s) by which
this metal, both ‘‘endogenous’’ and ‘‘exogenous,’’ can keep under appropriate
control the development of the inflammatory process.
Therefore, the present state of the art appears most encouraging for
the continuation of research to ascertain whether copper could represent a
truly innovative approach to the curing of human inflammatory diseases,
rheumatoid arthritis in primis.
Nevertheless, few serious attempts have been made, and none currently,
to study the use of copper in the therapy of such diseases. There
are several prejudicial barriers that keep copper out of both experimental
laboratories and the clinic: copper is generally thought to be far more toxic

Copper in Medicine and Personal Care 159
than it actually is (36) and, especially, the marketing of copper compounds
offers profits too low to be advantageous to business.
Nonetheless, we would like to stress that the pharmacological exploitation
of the anti-inflammatory and antiarthritic copper potential is
anything but an obsolete idea. On the other hand, rheumatoid arthritis
and related degenerative diseases that are found worldwide and are the
cause of great disability with high social and economic costs, still wait for
a pharmacological therapy that is both effective and safe. One can hope
for the continuation of research in this field.
ABBREVIATIONS
L taxonomic classification by Linné
R & P taxonomic classification by Ruiz and Pavon
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Roma: Gruppo Editoriale l’Espresso, 2004.
2. Sterpellone L. Dagli dei al DNA. Vol. 1. Chapters 1–6. Roma: Antonio Delfino
Editore, 1988.
3. Baines J, Málek J. Atlante dell’Antico Egitto. Novara: Istituto Geografico De
Agostini, 1985:36.
4. von Beckerath J. Handbuch der ägyptischen Königsnamen. MÄS 1984; 20:158.
5. David RA. The pyramid builders of ancient Egypt. In: Chapters V and VI.
London: Routledge and Kegan, 1986.
6. Rossi Osmida G. La scoperta della vanità. Archeo 1989; 58:62.
7. Guy E, Rachet MF. Dictionnaire de la Civilisation égyptienne. Paris: Librairie
Larousse, 1972:100,192.
8. Sorenson JRJ. Pharmacological activities of copper compounds. In: Berthon G, ed.
Handbook of metal–ligand interactions in biological fluids. Vol. 2. New York:
Marcel Dekker Inc., 1995:1128.
9. Windholz M, ed. The Merk index. 10th ed. Rahway: Merck & Co. Inc.,
1983:379.
10. Radicchi R. Civiltà sumero-akkadica. Chapters 4 and 5. Pisa: Giardini Editore,
1968.
11. Sterpellone L. Dagli dei al DNA. Vol. 2. Chapters 1–4. Roma: Antonio Delfino
Editore, 1988.
12. Capasso L. Aria al cervello. Archeo 1990; 63:121.
13. Cuory C. Mèdècine de l’Amerique precolombienne. Chapters 2,3, and 6. Paris:
Roger Dacosta, 1969.
14. Pravega D. La terapia nella medicina greca. Chapters 1–7. Pisa: Giardini
Editore, 1963.
15. Walker WR, Beveridge SJ, Whitehouse MW. Dermal copper drugs: the copper
bracelet and Cu(II) salicylate complexes. In: Rainsford KD, Brune K,

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Whitehouse MW, eds. Trace elements in the pathogenesis and treatment of
inflammation. Basel: Birkhäuser Verlag, 1981:359.
16. Lotz L-O, Weser U. Biological chemistry of copper compounds. In: Rainsford KD,
et al, eds. Copper and zinc in inflammatory and degenerative diseases. Chap. 3.
Dordrecht: Kluwer Academic Publisher, 1998.
17. Capasso L. I romani in farmacia. Archeo 1989; 57:54.
18. Ackerknecht EH. Therapeutics from the primitives to the 20th century. Chapters
I–VI. New York: Hafner Press, 1973.
19. Bonta IL. Microvascular lesions as target of anti-inflammatory and certain other
drugs. Acta Physiol Neerl 1969; 15:188.
20. Bynum WF, Browne EJ, Porter R, eds. Macmillan dictionary of the history of
science. New York: The Macmillan Press, 1981:449.
21. Vattimo G, Ferraris M, Marconi D (consultant authors). Le Garzantine: Filosofia.
2nd ed. Milano: Garzanti Libri s.p.a., 1999:1036.
22. Deuschle U, Weser U. Copper and inflammation. Prog Clin Biochem Med 1985;
2:97.
23. Hart EB, Steembock H, Waddel J, et al. Iron in nutrition. Vol. VII. Copper as a
supplement to iron for haemoglobin building in the rat. J Biol Chem 1928; 77:797.
24. Heilmeyer L, Stuwe G. Der Eisen-kupferantagonismus im Blutplasma beim
Infektionsgeschehen. Klin Wochenschr 1938; 17:925.
25. Wintrobe MM, Cartwright CE, Gubler CJ. Studies on the function and metabolism
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26. Linder MC. Biochemistry of copper. Chap. 4. New York: Plenum Press, 1991.
27. Sweeney SC. Alterations in tissues and serum ceruloplasmin concentration associated
with inflammation. J Dent Res 1967; 46:1171.
28. Lorber A, Cutler LS, Chang CC. Serum copper levels in rheumatoid arthritis: relationship
of elevated copper to protein alteration. Arthritis Rheum 1968; 11:65.
29. Karabelas DS. Copper metabolism in the adjuvant induced arthritic rat. Ph.D.
thesis. Ann Arbor: Michigan State University, 1972.
30. Rainsford KD. Adjuvant polyarthritis in rats: is this a satisfactory model for
screening anti-arthritic drugs? Agents Actions 1982; 12:452.
31. Sorenson JRJ. Copper chelates as possible active forms of the anti-arthritic
agents. J Med Chem 1976; 19:135.
32. Whitehouse MW. Ambivalent role of copper in inflammatory disorders. Agents
Actions 1976; 6:201.
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Therapeutic uses of trace elements. New York: Plenum Publishing Corporation,
1996:115.
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and control of inflammation. In: Handbook of metal–ligand interactions in biological
fluids. Berthon G, ed. Vol. 2. New York: Marcel Dekker Inc., 1995:886.
35. Sorenson JRJ. Copper complexes offer a physiological approach to treatment of
chronic diseases. In: Ellis GP, West GB, eds. Progress in medicinal chemistry.
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36. Medeiros DM, Wildman R, Liebes R. Metal metabolism and toxicities. In:
Massaro EJ, ed. Handbook of human toxicology. Chap. 3. Boca Raton: CRC
Press LLC, 1997.

9
The Role of Copper in Onset,
Development, and Control of
Acute and Chronic Inflammation
Roberto Milanino
Facoltà di Medicina e Chirurgia, Sezione di Farmacologia,
Dipartimento di Medicina e Salute Pubblica, Università di Verona,
Verona, Italy
INTRODUCTION
The involvement of ‘‘endogenous’’ copper in inflammation was discovered
in the second half of the last century when evidence was published that:
(i) total serum copper markedly increased in aseptic and septic acute inflammation,
such as turpentine edema, Staphylococcus aureus abscesses, and
typhoid vaccine injection (1953); (ii) total serum copper and ceruloplasmin
as well as the ceruloplasmin measured within the inflamed tissue significantly
increased in patients suffering from acute periodontal disease (1967);
(iii) total serum copper was found significantly elevated in rheumatoid
arthritic patients (1968), and in adjuvant-induced arthritis (AA) in the rat
(1972), still a very useful experimental model for that human disease (1–5).
It was, however, only in 1976, i.e., about two millennia after the first
This chapter, in part, was reprinted from Milanino R. Copper: an overview of the role of endogenous
and exogenous metal in the development and control of the inflammatory process. Rev
Environ Health 2006; 21:1–70, with permission.
161

162 Milanino
documented intuition by Celsus on this issue in pharmacology (6), that the
researchers’ attention again addressed the role of ‘‘exogenous’’ copper as a
potentially effective anti-inflammatory and antiarthritic drug. In fact, in
that year, two major papers were published.
The first one, due to the work of J.R.J. Sorenson (7), not only showed
that many subcutaneously injected copper(II) complexes or salts were very
active anti-inflammatory and antiarthritic agents, but also proposed that the
complexes extemporaneously formed in vivo with ‘‘endogenous’’ copper were
the real active forms of the most common and currently used antiarthritic drugs
(such as salicylate, aspirin, D-penicillamine, etc.). The above theory has actually
been the subject of a lively debate among medicinal chemists, coordination chemists,
and pharmacologists, and it is still an unresolved question.
The second, especially stimulating paper was published by M.W.
Whitehouse (8) in the same year. In that article, the author speculated on
the role of both ‘‘endogenous’’ and ‘‘exogenous’’ copper in inflammation,
based on the somewhat contradictory evidence known at that time:
In some circumstances, soluble copper preparations behave as
acute or chronic inflammatory (or irritant) agents.
Acute and chronic inflammations are both characterized by a significant
increase of total serum copper, which, in light of its irritating
potential, may be even regarded as a factor capable of worsening
the pathological process.
D-Penicillamine, a very well-known de-coppering agent that is still
now successfully used in the therapy of Wilson’s disease (9), is also
a valuable antiarthritic drug.
Pathological (jaundice and other liver diseases) or physiological
(pregnancy) events that cause total serum copper to remarkably
increase above normal levels, on the other hand, often brought
about spontaneous remission of rheumatoid arthritis.
Supplementation with copper using a number of folk remedies such as
Cu bangles, cider vinegar, shellfish, nuts, etc., as well as the treatment
with copper complexes or salts reported by Sorenson (7), seem to be or
actually are remarkably good anti-inflammatory/antiarthritic agents.
Thus, Whitehouse proposed that copper, either ‘‘endogenous’’ or
‘‘exogenous,’’ can be both injurious and beneficial, even at one and the same
time, acting in the organism as ‘‘inert,’’ ‘‘toxic,’’ or ‘‘pharmacoactive,’’
depending on the different physiological or pathological conditions characterizing
the organism itself.
The actual state of things was then clearly far from being settled, and
this intriguing issue induced a number of research teams to begin studying in
more detail the roles that ‘‘endogenous’’ and ‘‘exogenous’’ copper could
really have on the onset, development, and control of either acute or chronic
inflammation.

The Role of Copper in Inflammation 163
STUDIES ON COPPER-DEFICIENT, EXPERIMENTALLY
INFLAMED ANIMALS
In 1977, our research group set out the idea to study the development of
acute and chronic inflammatory processes in experimental animals made
deficient of copper by feeding them with a diet containing very low amounts
of this essential metallo-element. The rationale of the approach was based
on the assumption that inflammation developing in a copper-deprived
animal could perhaps give some valuable indication about what the prevailing
effect of copper on this kind of diseases was, i.e., a pro- or an
anti-inflammatory one. Before beginning the project, normal female and
male rats were kept on diets that, according to Owen and Hazelrig (10), were
either sufficient (10 ppm) or deficient (0.4 ppm) in copper for three months,
and then a complete toxicological examination of the animals was performed.
It resulted that the copper-deprived female rats had a reduced
ponderal growth compared with control animals. However, the major blood
cell and hematochemical serum markers were within normal range; moreover,
normal was also the condition of the numerous tissues, organs, and
apparatus, examined both macro- and microscopically (11). On the other
hand, the same copper-deficient dietary regimen was severely impairing
for male rats’ survival, and marked deviations from normality were found
evaluating many of the parameters mentioned above (unpublished observation).
This latter finding was subsequently confirmed by Fields and
coworkers (12), although it is still an unexplained phenomenon.
Acute Inflammation
First, the effect of copper depletion on the development of an aseptic acute
inflammatory process was examined in female rats kept on a 0.4 ppm Cucontaining
diet for 30 or 90 days, using the carrageenan-induced paw edema
(CPE) model (13). Between the 15th and the 20th day from the beginning of
the copper-deficient feeding, the concentration of serum copper was found dramatically
reduced, to about a mean 6% of the control values, and this level
remained constant throughout the whole experiment (Table 1, zero time).
The effects of the treatment on the development of the paw edema were, nonetheless,
totally different. In fact, after 30 days of copper deprivation the mean
swelling measured in the deficient group was comparable to that of the control,
whereas 90 days after the beginning of the experiment, the metallo-elementdeficient
animals developed an inflammatory reaction significantly greater than
that measured in the normally fed controls, which reached its maximum difference
(þ58%) about 20 hours after the subplantar irritant injection (Table 1).
The above proinflammatory effect of copper depletion was confirmed
in male rats by Denko and coworkers (14,15), who employed the same
experimental model (maximum increase of the foot edema þ31%), although
the deficient diet used was less severe in both copper content (0.6 ppm) and

164 Milanino
Table 1 Serum Copper Concentrations and Paw Edema Volumes After 30 and
90 Days of Normal (Cu ¼ 10 ppm) or Copper-Deficient (Cu ¼ 0.4 ppm) Feeding
Days of
diet
Hours after
challenge
Total serum copper
(mg/dL SD)
Paw edema volume
(plethysmographic
units SD)
Normal Deficient Normal Deficient
30 0 193.2 12.1 10.7 — —
3 185.9 20.3 11.9 14.1 22.2 3.4 21.3 2.6
5 179.2 20.6 10.1 13.8 24.1 3.5 22.9 3.9
20 270.3 22.3 a
91.7 18.1 a
15.4 3.6 14.5 3.8
90 0 190.4 21.0 12.9 11.4 — —
3 197.1 20.4 10.5 15.9 22.1 3.0 28.6 4.1 b
5 187.6 19.1 26.1 19.2 25.3 3.5 30.8 5.1 c
20 276.2 22.1 a
102.2 19.0 a
15.1 3.6 23.8 5.7 b
Note: Statistics—Student’s t test (data standard deviations; N ¼ 13 rats per group and per hour).
a P < 0.001; comparison versus the zero time values of either normally fed or deficient rats.
b P < 0.001; comparison versus the time-matched, normally fed animals.
c P < 0.010; comparison versus the time-matched, normally fed animals.
Source: The data reported in this table are the mean values of those obtained in an unpublished
preliminary experiment (N ¼ 3 rats per group) and those published in Ref. 13.
length of feeding (30 days). However, the data reported in Table 1 provided
other, relevant evidence. Firstly, the proinflammatory effect observed was
independent of the amount of total copper present in serum, but dependent
on the metallo-element-deficient length of the dietary intake. Secondly, at the
20th hour and in all tested groups, the acute inflammatory challenge promoted
a statistically significant increase of total serum copper concentration
that, later on, we demonstrated to be very highly correlated to the serum
ceruloplasmin level, as measured by the oxidase activity method (16). Interestingly,
this total serum copper increase was found to be far more dramatic in
the deficient rats (þ671% at day 30, and þ873% at day 90) when compared
with that measured in the control animals (þ45% at day 30, and þ40% at
day 90). Ceruloplasmin being a copper protein essentially synthesized and
secreted by the liver as an acute-phase reactant, and considering that it was
proposed to exert a protective role in inflammation, one could speculate that
this increase of circulating ceruloplasmin may represent the first evidence of a
main role for ‘‘endogenous’’ copper as a physiological agent acting in the
control of the development and remission of inflammatory process (14,17).
To better understand the soundness of the latter hypothesis, as well as
the fact that the proinflammatory effect of copper deficiency was independent
of total serum copper concentrations and, conversely, linked to the
amount of copper contained in the depleted diet and to the length of time
that the animals were exposed to it, further experiments were done. Two

The Role of Copper in Inflammation 165
different copper-deficient diets were considered (containing either 0.2 or
0.7 ppm of copper), fed to female rats for two different periods of time
(respectively, 30 and 150 days), in two models of aseptic carrageenaninduced
acute inflammation, i.e., the relatively mild CPE, and the more
distressing carrageenan-induced pleurisy (CP) (18).
The results obtained in the CPE test following the 0.2 ppm copperdepleted
diet regimen showed that the proinflammatory effect observed after
30 days of preliminary deficient feeding was not only very obvious (Table 2)
but also remarkably higher than that obtained in the previous experiment
(Table 1) (13). In fact, the average paw swellings measured three, five, and
20 hours after the challenge were of þ36% (0.4 ppm Cu diet for 90 days,
Table 1) versus a þ51% (0.2 ppm Cu diet for 30 days, Table 2); moreover,
the maximum enhancement of the local inflammatory reaction, which was
gauged at the 20th hour also in the 0.2 ppm diet-treated group, was of
about 95% (Table 2). A similar dramatic proinflammatory effect was promoted
as well by the 0.2 ppm copper-deficient diet given for 30 days in
the carrageenan-induced pleurisy model.
In the CP experimental disease, the intrathoracic injection of a sterile
carrageenan suspension caused the leakage, from the blood into the thoracic
cavity itself, of an inflammatory exudate composed by a fluid fraction in
which a large amount (tens of millions) of ‘‘inflammatory’’ cells (mainly
Table 2 Serum Copper Concentrations and Paw Edema (CPE) or Exudate (CP)
Volumes After 30 Days of 0.2 ppm Copper-Deficient Feeding (Control Diet:
Cu ¼ 10 ppm)
Inflammatory
model
Hours after
challenge
Total serum copper
(md/dL SD)
Paw edema or
exudate volumes
(plethysmografic
units or mL SD)
Normal Deficient Normal Deficient
CPE 0 171.2 22.5 4.6 7.2 0 0
3 167.2 29.6 7.3 7.6 18.2 1.6 25.4 3.7 b
5 163.5 30.1 5.8 4.3 24.3 2.1 28.9 1.5 b
22 291.3 29.4 a
24.9 26.8 10.1 3.3 19.7 3.1 b
CP 0 171.4 29.1 7.9 5.9 0 0
6 173.8 26.1 6.7 3.7 0.83 0.3 1.64 0.4 b
22 248.8 24.2 a
12.1 9.4 — —
Note: Statistics—Student’s t test (data standard deviations; N ¼ 13 rats per group and per hour).
a
P < 0.001; comparison versus zero time values (CPE or CP), of either normally fed or
deficient rats.
b
P < 0.001; comparison versus time-matched, normally fed animals.
Source: The data reported in this table are the mean values of those obtained in an unpublished
preliminary experiment (N ¼ 3 rats per group) and those published in Ref. 18.

166 Milanino
neutrophils) were present. This inflammatory reaction (that is routinely
scored six hours after the challenge) was found to be remarkably higher in
the copper-deprived rats compared with the response of the normally fed
pleuritic animals (þ98%, Table 2). Nevertheless, the increase of the total
exudate volume was accompanied by a proportional rise in its cellular component,
which, in turn, seemed to suggest that the anti-inflammatory activity
of ‘‘endogenous’’ copper might have little effect on the overall process of
leukocyte migration, at least in conditions of copper deficiency and, in this
particular model, of acute inflammation. However, another evidence needs to
be stressed. The severity of the 0.2 ppm-induced copper deficiency seemed
to have reduced the body stores of the metal to such an extent as to prevent
the inflammatory process from triggering the statistically significant increase
of total serum copper (Table 2). Such an increase is seen, as a rule, in
normally fed rats, and was reported also in the copper-depleted, inflamed
animals examined in the previous experiment (Tables 1 and 2) (13).
When the above acute inflammations (i.e., CPE and CP) were induced
in rats fed a 0.7 ppm Cu diet for about five months, no significant differences
were measured in either the paw edema or the total exudate volumes
between normally fed and copper-deficient animals (Table 3) (18).
Table 3 Serum Copper Concentrations and Paw Edema (CPE) or Exudate (CP)
Volumes After 150 Days of 0.7 ppm Copper-Deficient Feeding (Control Diet:
Cu ¼ 10 ppm)
Inflammatory
model
Hours after
challenge
Total serum copper
(md/dL SD)
Paw edema or
exudate volumes
(plethysmografic units
or mL SD)
Normal Deficient Normal Deficient
CPE 0 172.3 27.9 21.5 20.8 0 0
3 — — 18.9 6.0 20.3 5.7
5 — — 29.3 8.2 27.9 8.4
22 250.4 31.8 a
70.1 89.9 13.8 5.1 12.7 4.9
CP 0 171.4 29.1 21.3 29.1 0 0
6 — — 1.0 0.2 1.1 0.4
22 285.3 33.9 a
89.6 86.4 b
— —
Note: Statistics—Student’s t test (data standard deviations; N ¼ 13 rats per group and per hour).
a
P < 0.001; comparison versus the zero time values (CPE or CP), of either normally fed or deficient
rats.
b
P < 0.050; comparison versus the zero time values (CPE or CP), of either normally fed or deficient
rats.
Source: The data reported in this table are the mean values of those obtained in an unpublished
preliminary experiment (N ¼ 3 rats per group) and those published in Ref. 18.

The Role of Copper in Inflammation 167
The determination of total serum copper levels (made at zero time
and 20 hours after the challenge) showed that this parameter increased, as
expected, in the inflamed controls, but its rise was far less significant in
the inflamed-depleted animals. However, this latter and unpredicted result
is not considered to be a biologically significant one since the single means
measured were characterized by extremely high standard deviation values
(Table 3) (18).
The above-summarized data lead to suggest the following proposals:
In rats, the induction of a copper-deficiency status is a condition
that promotes a very significant enhancement of the acute inflammatory
reaction, which, in turn, speaks in favor of an anti- rather
than a proinflammatory role of ‘‘endogenous’’ copper.
The observation that the amount of copper left in the diet and/
or the length of the deprived feeding are able to influence the
reported phenomenon seems to indicate a sort of dose- and
time-dependent effect of copper deficiency, testifying that this
phenomenon is a real and not a casual event. It must be stressed
that this conclusion has been confirmed in a subsequent study by
Kishore and coworkers (19).
The rats not dramatically depleted of copper (i.e., those that underwent
feeding with a diet containing 0.4 ppm of the metallo-element)
react to the challenge by secreting relatively striking amounts of
ceruloplasmin into the blood from the liver. Considering that this
protein appears to exert an anti-inflammatory action in vivo (14,20),
this evidence seems to further support the hypothesis of a natural
defensive role of ‘‘endogenous’’ copper in inflammation.
Finally, the findings just mentioned led us to speculate that the
appearance of the enhanced acute inflammatory reaction promoted
by the deficiency of copper could strictly depend on the amount of
copper still present in the liver (21). Later on, this hypothesis found
its experimental demonstration in male rats fed a 0.5 ppm Cu for
20, 40, or 60 days and then tested in the CPE model (19). Interestingly,
in the same paper the author showed that not only the paw
edema in copper-deficient animals was highly and negatively correlated
to the concentration of copper in the liver, but also that
the correlation with liver Cu,Zn superoxide dismutase (SOD 1)
activity was inconsistent (19). Conversely, the activity of SOD 1
in total blood cells of copper-deprived rats (fed a diet containing
0.2 ppm of copper, for one month) was found to be reduced to
about 50% of the control values; similar results were also obtained
in copper-deficient pigs (22,23). These two last observations directed
the attention to the role that superoxide radicals play in the
development of the inflammatory process in vivo, and, in turn, in

168 Milanino
the scavenging activity of many copper compounds other than the
SOD 1 itself (24,25). Thus, the above information led us to speculate
that ‘‘endogenous’’ copper could control the inflammation also by
regulating the superoxide metabolism, and, as a consequence, its
deficiency may, at least in part, justify the exacerbation of the acute
inflammatory reaction observed in the laboratory animal (18).
Chronic Inflammation
The effects of dietary copper deficiency on experimental chronic inflammation
were first studied in the adjuvant arthritis of the female rat, after
60 days of feeding the animals with either a copper-depleted (Cu 0.4 ppm)
or a control (Cu 10 ppm) diet, before carrying out a tail-intradermal injection
of heat-killed Mycobacterium butyricum finely suspended in liquid
paraffin (complete adjuvant) (26). Note that the copper-deficient feeding
was continued throughout the experiment (26).
As expected, in the normally fed rats the systemic reaction to the challenge
began to become evident macroscopically at about 12–14 days after
the inoculum and reached its maximum development between the 18th
and the 28th day (Fig. 1, upper panel). Together with a marked loss of
body weight, the other major pathological signs [that represent the basis
of the arthritic score routinely used to assess disease severity, the maximum
theoretical value of which is 31 (5,26)] showed: (i) a dramatic swelling of the
paws, particularly the hind ones; (ii) the appearance of numerous arthritic
nodules randomly disseminated in the tail; and, especially, (iii) a very deep
bone and cartilage degradation at the joint levels (chiefly the hind tibiotarsals),
very clearly documented at both X-ray and histological examination,
of which a severe joint stiffness and, in turn, a dramatic impairment
of the deambulation ability were the consequence. In our experiment, in this
group of normally fed animals, the highest arthritic score (29.4) was measured
at day 21 (Fig. 1, upper panel) (26). At the same time, a progressive
increase of total serum copper concentration was observed, which also
reached its maximum (þ82%) 21 days after complete adjuvant injection
(Fig. 1, lower panel) (26). On the contrary and rather surprisingly, the
copper-deficient animals did not react to complete adjuvant injection, and
the systemic response expected was almost totally abolished by dietary
copper depletion. In fact, only a barely visible swelling of the hind paws
was observed 21 days after the challenge (highest arthritic score 4.3; Fig. 1,
upper panel), and the X-ray and histological examinations showed that the
joint status of these copper-deficient and complete adjuvant-injected rats
appeared to be almost fully normal (26). Moreover, throughout the experiment
the total serum copper concentration, albeit extremely low, remained
constant ranging between 10 and 15 mg/dL (Fig. 1, lower panel) (26). The
fact that the diet-induced copper deficiency was a condition able to strongly

The Role of Copper in Inflammation 169
Arthritic
score
Serum Cu
(µg/dL)
35
28
21
14
7
0
350
280
210
140
70
0
*
0 7 14 21 28
Days after the inoculum
*
* * * *
0 7 14 21 28
Days after the inoculum
Cu-normal
Cu-deficient
Figure 1 Development of the adjuvant arthritis (arthritic score) and increases of
total serum copper concentrations in normally fed and dietary Cu-depleted rats.
Arthritic score (upper panel) and total serum copper (lower panel) in normally fed
and copper-deficient adjuvant-arthritic rats. Source: From Refs. 11 and 26.
inhibit the normal development of the AA of the rat was confirmed by other
authors (27).
Previous reports have shown that copper is involved in the prostaglandin
biosynthesis, and, in particular, that it seems to increase the conversion
of arachidonic acid to prostaglandin F2a (an anti-inflammatory mediator),
and to reduce the production of the prostaglandin E2 (a proinflammatory
product of the arachidonic acid cascade) (28–31). In our experiment (26),
we showed that the lung prostaglandin-synthetase activity was the same in
*
Cu-normal
Cu-deficient

170 Milanino
normal and copper-depleted nonarthritic animals. Moreover, no difference
was observed in the reactivity to exogenous prostaglandin E2 or F2a added
to strips of rat stomach fundus or colon from nonarthritic copper-deficient,
compared with nonarthritic normally fed animals. Thus, the above data
seemed to exclude the influence of copper depletion on prostaglandin
synthesis pathway as a possible mechanism explaining the observed phenomenon.
On the other hand, at that time it was already known that a lower
immunological reactivity (as measured by cytotoxic anaphylactic reaction,
phagocytic, and serum bactericidial activities) was promoted by copper
deficiency in the rat (32). Consequently, considering that in the development
of the adjuvant-arthritis model a normal response of the immune system is
strictly required, we proposed that the protective effect of copper deficiency
on the ‘‘normal’’ development of the AA was an epiphenomenon secondary
to an impairment of the immune function (5,26). This hypothesis was
confirmed later on by Kishore and coworkers (33), who showed that the
copper-deficient male rats (Cu in the diet 0.5 ppm; length of feeding 40 days)
were actually in a state of apparent immunosuppression as demonstrated by
impaired ex vivo responsiveness to the T-cell-dependent contact-sensitizing
antigen oxazolone and diminished capacity to respond to the T-cellindependent
antigen Type III pneumococcal polysaccharide stimulation.
LABORATORY ANIMALS: STUDIES ON ‘‘ENDOGENOUS’’
COPPER METABOLISM IN ACUTE AND
CHRONIC INFLAMMATION
Illnesses always promote more or less remarkable, and sometimes dramatic,
changes of the whole-body homeostatic mechanisms that come into play
with the aim of self-defense, or may be ‘‘side effects’’ of the disease itself,
which could even contribute to worsening the condition. This was, actually,
the theoretical question posed by Whitehouse (8) speculating on the possible
‘‘inert,’’ ‘‘toxic,’’ or ‘‘pharmacoactive’’ role of ‘‘endogenous’’ copper in the
development and control of inflammation. Thus, in light of the ascertained
proinflammatory effect of nutritional copper deficiency on the development
of the acute processes, it seemed reasonable to conduct more detailed studies
on the changes in ‘‘endogenous’’ copper metabolism induced by the inflammatory
process in laboratory animals. Some of the most significant results
obtained are summarized in Tables 4 (and references therein; acute inflammation)
and 5 (and references therein; chronic inflammation).
Status of Copper in Blood and Urine
Since the early reports, many different studies carried out in different animal
species have shown that the acute inflammatory process, regardless of the
noxa used to promote it, is characterized by a significant increase of total

The Role of Copper in Inflammation 171
Table 4 Experimental Animals: Examples of the Changes in Copper Status
Induced by Acute Inflammation in Some Relevant Body Compartments
Species Disease
Total
serum
Blood
cells Liver
Kidney
Inflamed
area References
Rat Turpentine edema I U — — — 1
Radiation injury U — — — — 34
Turpentine abscesses I — I — — 35
Femur fracture — — D — — 36
CP I I U U — 37
CP I U U — P 38
CPO I U U — I 38
Rabbit Turpentine edema I — — — — 39
Ocular inflammation I — — — I 40
Ocular inflammation — — — — I 41
Dog Turpentine abscesses I — — — — 42
Guinea pig Turpentine abscesses I — — — — 43
Mouse Collagenase injection I — — — — 2
Abbreviations: I, significantly increased; U, unchanged; D, significantly decreased; P, present in
the inflammatory exudate.
serum (or plasma) copper concentration (Table 4) (2,3,34–43). An exception
was apparently represented by the effect of radiation injury in the rat (34).
However, this result failed to be confirmed by more recent work, which
showed a remarkable increase of serum copper, even in that experimental
model of inflammation (44,45). Also, chronic inflammation induced in
the experimental animals typically brings forth a remarkable rise of total
serum (or plasma) copper (Table 5), albeit an occasional report showed
that, in dogs chronically suffering from nonexperimentally induced dermatitis
or anal gland fistula, these diseases did not cause significant changes in
metallo-element status in the serum (4,33,42,46–51).
As a general rule, the inflammation-induced increase of total serum
copper is accompanied by a parallel increase of serum ceruloplasmin
concentration, and, as noted before, these two parameters characterizing
serum copper status are highly and statistically significantly correlated in
normal as well as acutely and chronically inflamed animals (4,16,52,53).
Moreover, it has been shown in hamsters that transcription of ceruloplasmin
mRNA increases within three hours of induction of inflammation by turpentine
injection, reaching a peak 2.5-fold above normal at 12–18 hours (54).
Thus, at least in animals fed a diet containing standard amounts of copper,
the total serum (or plasma) copper measured during inflammation appears
to represent an extremely reliable index of circulating ceruloplasmin levels,
which, in turn, belies an old report according to which the human inflammatory
process (rheumatoid arthritis) is distinguished by a selective rise of the

172 Milanino
Table 5 Experimental Animals: Examples of the Changes in Copper Status Induced
by Chronic Inflammation in Some Relevant Body Compartments
Species Disease
Total
serum
Blood
cells Liver Kidney
Inflamed
area References
Rat AA I — I D — 4
AA I — I — — 33
AA I — I — — 46
Chronic
I — I — — 47
inflammations
AA I I I D I 48
AA I U I — — 49
AA I — I U — 50
Dog S. aureus-induced
arthritis
I — — — — 51
Chronic otitis I — — — — 49
Abbreviations: AA, adjuvant-induced arthritis; I, significantly increased; U, unchanged; D, decreased.
non-ceruloplasmin-bound fraction of serum copper (1). This latter claim was
subsequently further disproved by Freeman and O’Callaghan (55), who,
directly measuring (following column fractionation) the non-ceruloplasmin-bound
copper in the serum of normal and inflamed animals and
humans, showed that this fraction of circulating metal undergoes only negligible
variations during both adjuvant arthritis in the rat as well as human
rheumatoid arthritis.
Another issue is worth emphasizing in view of its relevance in the
studies on ‘‘endogenous’’ copper involvement in human inflammatory diseases.
Although the above-reported increases of circulating copper and/or
ceruloplasmin are important markers in revealing the existence of an inflammatory
state [unless, for example, pregnancy is present, or the subject is
undergoing steroid therapy (56)], the data obtained during the past years
in our laboratory, studying over 1200 inflamed rats, show that serum or
plasma copper as well as ceruloplasmin concentrations are not directly correlated
with the actual severity of the pathological condition examined, and
cannot even discriminate between acute and chronic processes. In fact, it
was found that different acute nonseptic inflammations (e.g., rat CPE and
CP), which developed in very similar manner, caused remarkably variable
increases in the above parameters (57,58). A great variation in increased
total plasma copper concentration was also reported among adjuvantarthritic
rats regardless of illness severity level (26,48). In particular, the
average increase in total plasma copper in animals, which manifested experimental
disease to a mild degree, was of about 54%, and that measured in
rats bearing a fully expressed pathology was approximately 60% (46). Therefore,
hypercupremia that characterizes any inflammatory reactions appears

The Role of Copper in Inflammation 173
to be a sort of ‘‘all or nothing’’ phenomenon that has indeed little value as a
marker of disease seriousness.
To conclude the analysis on copper status in blood of acutely or
chronically inflamed animals, the erythrocyte compartment remains to be
considered. A number of contradictory data have been reported on this
issue. In fact, according to some authors neither acute nor chronic inflammatory
processes changed the concentration of copper in the erythrocytes,
whereas an increase of this fraction of total blood copper was reported in
rats with carrageenan-induced pleurisy and adjuvant arthritis (3,37,38,
48,49). However, the increase of erythrocyte copper measured in the arthritic
rats (48), although statistically significant at day 3 and 21 after challenge,
was small and probably did not have major biological relevance. Moreover,
as ascertained later on studying the same parameter in human rheumatoid
arthritis (59), the rise of copper in the red cells of chronically inflamed subjects
was, most likely, a secondary phenomenon on which the significant
decrease in erythrocyte volume had a not negligible influence.
Finally, very few and contradictory data exist on the urinary copper
excretion in acutely and chronically inflamed rats. For example, the metal
concentration in the urine was found to increase in femur-fractured animals,
whereas the challenge with complete adjuvant did not modify this
parameter (36,49). Therefore, at least as the experimental models are concerned,
it is not possible to draw any conclusion on this topic on the basis
of existing evidence.
Status of Copper in Solid Tissues and Inflamed Areas
Before getting into a detailed discussion of inflammation-induced changes
in copper status in some solid tissue involved in onset, development, and
control of the inflammatory processes, a preliminary issue deserves attention.
In most, if not all, papers dealing with copper and inflammation, the
amount of the metallo-element measured in the different body compartments
is exclusively reported as concentration values. Nevertheless, in some
instances this could be a misleading index. As a matter of fact, we studied
the concentration and total amount of liver copper during a daily ninehour
period in normal rats, i.e., between nine hours and 30 minutes and
18 hours and 30 minutes (Table 6) (60).
The results reported in Table 6 clearly show that during the period of
time considered, an apparent statistically significant increase in liver copper
concentration occurred. However, this phenomenon was essentially due to a
parallel and progressive decrease of liver weight (possibly caused to different
states of liver hydration and/or glycogen content); conversely, the total
amount of the metal measured in this compartment remained constant
throughout the experiment (60). This physiological phenomenon strongly
suggests: (i) the evaluation of copper concentration and total amount, as well

174 Milanino
Table 6 Daily Changes in Hepatic Copper Concentration and Total Amount in
Normal Female Rats
Time
(hours)
Total serum
Cu (mg/dL)
Liver weight
(g)
Liver copper
Cu (mg/g)
Total liver
Cu (mg)
0 151.3 6.6 4.31 33.03
3 þ8% 4% þ5% þ2%
6 þ0% 12% a
þ16% b
þ5%
9 þ3 21% b
þ25% b
þ3%
Note: Statistics—Student’s t test.
a
P < 0.05.
b
P < 0.01.
Source: From Ref. 60.
as (ii) the routine use of a control group (‘‘time controls’’) should be utilized
in every experiment, and these rats should be killed at the same time as those
of each treated group, thus ensuring that the results obtained and their discussion
could have a better chance of being fully reliable (60). Actually, we
strictly followed this rule in all the experiments carried out in our laboratory.
As shown in Tables 4 and 5, the liver is the tissue in which copper
status has been most frequently studied in inflamed animals. We already
know that the liver is the major organ responsible for ceruloplasmin synthesis
and secretion into blood (17,56). Then, in the acute processes, the first
striking evidence is that, in spite of the remarkably increased production and
release of this protein induced by inflammation, the level of total hepatic
copper does not decrease (37,38). The femur fracture in the rat is, perhaps,
the only model of an acute inflammatory process in which a reduced liver
copper concentration was reported (36); whether or not this isolated result
depends on the peculiarity of this important traumatic condition is unknown.
On the other hand, when chronically inflamed animals were studied, the
hepatic level of copper was always found to be significantly increased, both
as concentration and total amount values (4,33,46–50). Moreover, we observed
in the adjuvant-arthritic rat that a rise of hepatic copper level preceded
the appearance of any visible signs of the disease, and, afterwards was found
to be directly proportional to the manifested severity of the pathology
(46,61). In greater detail, liver copper total amounts were statistically significantly
increased above control values three days after the challenge (þ15%),
i.e., during the asymptomatic phase (46). Furthermore, when the disease
reached its complete development (i.e., at day 14, 21, and 30), the average
hepatic copper rise was of about þ61% in the severely arthritic rats, but only
of about þ31% in those animals that manifested the pathology to a mild
degree, these two data being significantly different in the cross Student’s t test
evaluation (48). Thus, according to our results and in contrast with total
serum copper and ceruloplasmin concentrations, the hepatic metal level

The Role of Copper in Inflammation 175
status appears to be a consistent index of the overall importance of the
inflammatory process, being able to clearly discriminate among acutely,
and chronically weakly or seriously affected rats.
The kidneys, together with the red blood cells, the liver, and to some
extent the plasma, are compartments in which copper seems to be easily
exchangeable and thus available to be moved to other tissues according to
actual body needs (61). As a consequence, the study of copper status in
the kidneys could provide useful information to better understand the effect
of inflammation on this ‘‘endogenous’’ metal metabolism. Unfortunately,
very few data are available, especially on the acutely inflamed animals, in
which the renal copper level was found not to be influenced by the development
of the carrageenan-induced pleurisy of the rat (37). On the contrary,
the chronic inflammatory process appeared to cause a significant decrease
of kidney copper levels, although other authors failed to confirm this evidence
(4,48–50). One of the above papers described that, like hepatic copper
increases, the decreases of both concentration and total amount of copper in
kidneys were not only statistically significant during the asymptomatic
phase of the rat AA (day 3), but also dependent on the seriousness of disease
development, and reached their maximum level in the severely arthritic rats
30 days after complete-adjuvant injection ( 48% compared to the total
metal amount measured in the time- and age-matched, healthy controls)
(48). Whether or not the decrease in total copper amount observed in the
kidneys (which, differently from the liver, cannot be promptly supplied by
metallo-element from the digestive tract) may be the expression of the need
for a physiological mobilization of copper towards extrarenal biological
pathways more directly involved in the control of the inflammatory process
is presently unknown.
Occasionally, but limited to the rat AA model, the concentration of
copper in compartments other than blood, liver, and kidney has been studied.
Thus, copper levels were found to be increased in the brain, stomach,
bone, and pancreas, while, according to the same paper, they were unchanged
in the heart and skeletal muscle (50). Other authors, however, found
the concentration of this metal unaffected in the femur and in the brain
(49,62,63). An increase of copper concentration and total amount was measured
in the spleen of the adjuvant-arthritic rat, being once again directly
related to the seriousness in the development of pathology (46). However,
these increases actually may bear, per se, an uncertain biological meaning,
since they were found to be mainly dependent on the increases of spleen
weight and total serum copper levels, both of which were induced by the
experimental pathology itself (48).
Although very rarely evaluated, the analysis of copper status within
inflamed fluids and tissues certainly deserves special attention. The evidence
so far available shows that copper concentration in acute inflammations
(Table 4) was found to be higher, compared with the noninflamed fluids

176 Milanino
or tissues, in the inflamed and untreated aqueous humor of endotoxininjected
rabbits, in the intrathoracic exudate caused by the injection of a
sterile carrageenan suspension in the rat (in this instance, due to technical
reasons, the comparison with the extremely small amount of fluid present
in the thoracic cavity of noninjected animals was not done), and in the
carrageenan-injected, inflamed rat paw (sectioned at the level of the tibiotarsal
joint) (38,40,41); in the last case a significant inflammation-induced
copper increase was observed, considering both the concentration and the
total amount of metallo-element. Copper concentration was, instead, found
unchanged in the colon of rats in which an experimental untreated colitis
was induced (64). On the other hand, the intravenous (IV) injection of 67 Culabeled
porphyrins in male rats showed a very remarkable uptake of the
radioactive compound not only in the liver and kidneys but also in neoplastic
tissue, when present and wherever localized in the animal body (65). In
addition, the subcutaneous (sc) treatment with 64 Cu-labeled D-penicillamine
of rats with an experimental sponge granuloma evidenced a selective accumulation
of the radioactive metal within both the implanted sponge and
the capsule that had surrounded it as a consequence of the organism’s reaction
(66). Also in the AA rat model (Table 5), the inflamed hind paws (again
sectioned at the tibio-tarsal joint) showed a statistically significant rise in
copper concentration (and total amount), already evident during the asymptomatic
phase (i.e., seven days after the challenge), and became dramatic
when the disease reached its symptomatic stage (48,62,63). Like previously
described for the liver and the kidneys, when the symptomatic phase was
evaluated specifically, the copper status of the hind paws was clearly able
to discriminate between slightly and severely affected animals (Table 7).
Concerning the increased amounts of copper in the carrageenaninjected
rat paw, a further point is worth emphasizing. The onset of the
Table 7 Hind Paws Copper Status During the Symptomatic Phase of the
Complete-Adjuvant-Induced Arthritis of the Rat
Days after the
inoculum
14 þ26% a
21 þ38% a
30 þ32% a
Mild disease Severe disease
Cu (mg/g) Cu total (mg) Cu (mg/g) Cu total (mg)
þ92% a
þ50% a
þ47% a
þ39% a,b
þ136% a,b
þ106% a,b
þ160% a,c
þ183% a,c
þ260% a,c
Note: Statistics—Student’s t test.
a P < 0.01 versus time- and age-matched noninflamed controls.
b P < 0.01 versus copper concentration of time- and age-matched mild-disease group.
c P < 0.01 versus copper total micrograms of time- and age-matched mild-disease group.
Source: The data reported in the table are converted to percentage increases from the original
values published in Ref. 48.

The Role of Copper in Inflammation 177
acute inflammation is characterized by progressive leakage from the local
vascular bed, of an inflammatory exudate made by a plasma-proteins-rich
fluid (containing also ceruloplasmin) in which a great number of inflammatory
cells (mainly leukocytes) are present (67). This phenomenon induces
the formation of an edema that, when carrageenan is used as inflammatory
agent, is already measurable one hour after the challenge, and, in most
instances, reaches its highest values between three and about 4–8 hours later
(68,69). Subsequently, the liquid fraction of the exudate is rapidly reabsorbed,
whereas the cell fraction remains in situ for several days to carry out the
repair and remission processes (67). If the data reported in Table 8 are taken
into account, it clearly emerges that the statistically significant increase of
paw-copper concentration precedes the hours in which the characteristic rise
of total plasma copper (and ceruloplasmin) is measured. Moreover, the paw
copper is still significantly elevated above control values 96 and 144 hours
after the challenge, i.e., when the edema has almost entirely disappeared
and the total plasma copper returned to normal. The same is also observed
if the total amounts of paw copper are considered (data not shown).
In the chronic processes, the edema present in the affected animals is
mainly formed by a solid tissue made of different inflammatory cell types
(such as leukocytes, lymphocytes, macrophages, fibroblasts, epithelioid cells,
etc.) together with local necrotic cells and newly formed fibrous as well as
synovial tissues; but, in contrast with acute inflammation, the serum-plasmaderived
fluid component is much less prominent (5,67). Evaluating the status
of copper in the hind inflamed paws of the arthritic rats, a scenario is observed
Table 8 Percentages of Concentration Changes in Plasma and Paw Tissue Copper
in Carrageenan-Inflamed Rats
Time after the
challenge (hours)
Edema
weight (g)
Total plasma
copper (mg/g)
Paw copper
(mg/g)
0 0 0 0
1 0.38 a
4 þ12 a
3 1.04 b
2 þ20 b
5 1.19 b
3 þ19 b
24 0.51 b
þ74 b
þ21 b
48 0.44 a
þ63 b
þ26 b
72 0.41 a
þ55 b
þ25 b
96 0.23 þ8 þ25 b
144 0.16 2 þ21 b
Note: Statistics—Student’s t test versus the time- and age-matched controls.
a P < 0.05.
b P < 0.01.
Source: From Refs. 37, 38, and 60.

178 Milanino
similar to that described in the case of the acutely inflamed animals. In
particular, as pointed out before, a significant increase of copper concentration
and total amount are measured during the asymptomatic phase of the
experimental disease (day 7; þ14%, P < 0.01), and, later on, these same parameters
appeared to be very reliable markers of the severity in experimental illness
(Table 7) (48,62,63). Interestingly, the whole blood copper amounts
(calculated on the basis of body weight, according to the Altman and Dittmer
formula) were found comparably higher throughout the experiment, being
insignificantly different among them in the asymptomatic, slightly and seriously
affected rats (48,70). Thus, all the evidence summarized above seems
to suggest that the increase of copper, quantified in the acutely and chronically
inflamed rat paw tissue, is not merely a consequence of the increased
plasma copper levels, but, rather, the expression of a true and selective metal
accumulation in this area. This event can, in turn, be seen as a signal revealing
an increased local need for copper, which could have been induced by the
reaction that the body promoted to counteract the inflammatory noxa.
Finally, taking into account all the organism’s compartments in which
acute or chronic inflammation-induced significant changes of the total copper
amount were measured [i.e., the whole blood, liver, kidney, spleen, inflamed
pleural exudates, and inflame paw (37,38,48,60,62,63)], we recalculated the
comprehensive variations of the rat body copper content brought about by
the inflammatory processes examined. The data obtained (Fig. 2) clearly show
that: (i) both acute and chronic inflammatory processes induced an overall
increase amount of the metallo-element in the organism; (ii) although the
total whole blood copper content (which we know to be raised to comparable
extents as response to any sort of inflammation) and the total content of kidney
copper (that, as previously reported, was reduced in the AA of the rat)
were taken into account in the calculation, the net accumulation of body
copper reported in Figure 2 was found directly proportional to the severity of
the experimental pathology studied. Importantly, the above-mentioned accumulation
appeared to occur without depleting other compartments of the
metal, such as the bone and the muscle, which are known to hold high
amounts of copper (49,50,61). Finally, considering that the healthy adult rat
contains about 2 mg/g of body weight of copper (71), the total copper content
of the control animals that we used for our experiments ranged between 250
and 420 mg. Thus, the statistically and biologically significant inflammationinduced
increases of the metal reported in Figure 2 ranged between 5% and
7% in the acutely inflamed rats, and may even approximate 25% in the case
of the severely affected arthritic animals. In conclusion, as commented for
the inflamed tissue, the above observation seems to suggest that the inflammatory
challenge induced also a whole-body increased demand for copper,
to be used in the onset and development of the physiological regulatory
mechanisms devoted to keep the inflammatory process under a
proper control.

The Role of Copper in Inflammation 179
Accumulation
of body copper
(Total µg)
56
42
28
14
0
a
Acute
inflamm.
a
Chronic
inflamm.
Asymptom.
Figure 2 Comprehensive copper accumulation in some rat body compartments relevant
to the inflammatory process. Accumulation (expressed as the difference of total
micrograms between inflamed and noninflamed animals) of total copper measured in
the body compartments in which significant inflammation-induced variations of
copper status were measured. The bars represent the net body increments of the
metal promoted by the different experimental acute and chronic inflammatory processes
studied in various rat models. Statistics (Student’s t test): a, P < 0.001 versus
age-matched non-inflamed controls; b, P < 0.001 versus age-matched asymptomatic
arthritic rats; c, P < 0.001 versus age-matched mild-disease arthritic rats. Source:
From Refs. 37, 38, 48, 62, and 63.
HUMAN SUBJECTS: STUDIES ON ‘‘ENDOGENOUS’’
COPPER METABOLISM IN ACUTE AND CHRONIC
INFLAMMATIONS, WITH A PARTICULAR REFERENCE
TO RHEUMATOID ARTHRITIS
The inflammation-induced changes of ‘‘endogenous’’ copper metabolism
were also frequently studied in humans, focusing especially, though not
exclusively, on rheumatoid arthritis (RA) (Table 9, and references therein).
Due to obvious ethical reasons, the experimental approach to this problem
was restricted to the body compartments in which an examination by nonor
mini-invasive techniques was possible, i.e., in the plasma, red blood cells,
urine, and, in few instances, the inflamed areas. The foremost aims of these
a,b
Chronic
inflamm.
Mild dis.
a,b,c
Chronic
inflamm.
Severe dis.

180 Milanino
Table 9 Examples of the Changes of Copper Status Induced by Some Acute (A)
and Chronic Inflammatory Diseases in Man (Cp ¼ Ceruloplasmin)
Disease
Serum
total Cu
Serum
Cp
Blood
cells
24 hours
urine
Inflamed
area References
Pneumonia (A) I — U — — 72
Periodontal disease (A) I I — — I 2
Tonsillitis (A) I — — — — 73
Sandfly fever virus (A) I — — D — 74
RA I U — — — 3
RA I — — — I 75
RA U — — — U 76
RA U — — — — 77
Ankylosing spondylitis I — — — — 78
RA I — — I — 79
RA I I — — I 80
RA I I U — — 81
RA I I — — — 82
RA (juvenile) I I — — — 83
RA I — — — I 84
RA I — — — — 85
RA I — I U — 86
Psoriatic arthritis I — U — — 87
RA I I U U — 88
RA (juvenile) I — — — — 89
RA I I I U — 59
Abbreviations: I, significantly increased; U, unchanged; D, decreased; RA, human rheumatoid
arthritis.
researchers were: (i) to verify the hypothesis that a marginal copper deficiency
may be a contributory factor in the etiology of rheumatoid arthritis;
(ii) to validate the assumption that the development of a rheumatoid process
could, in time, induce a depletion of the body copper stores; and (iii) to
evaluate whether or not the copper status in serum could be taken as a
reliable index of the severity of the chronic pathology studied (90–93). In
particular, an experimental support to either or both the above (i) and (ii) theoretical
propositions would offer a rational justification for the use of the
‘‘exogenous’’ copper administration as an important metal re-equilibrating
treatment, able to favor the amelioration, if not the resolution, of the
rheumatoid condition.
In humans, the existence of infectious, immune (or autoimmune), and,
in general, any acute or chronic disease with a relevant inflammatory component
(cancer included), is typically characterized by a significant increase of
total serum (or plasma) copper and ceruloplasmin concentrations, these
two parameters being highly and significantly correlated (59,80–83,94). As

The Role of Copper in Inflammation 181
previously noted while discussing the animal models of inflammation, in the
case of rheumatoid arthritis, in spite of the very high correlation existing
between serum copper and ceruloplasmin levels, some authors found that
the increase of the serum metal was essentially determined by a selective rise
of the non-Cp-bound fraction of this parameter (3). This claim was proposed
on the basis of the data obtained indirectly measuring the non-Cp-bound
copper, i.e., calculating this value by subtracting from the total serum copper
concentration the amount of the metal supposed to be carried by the ceruloplasmin
itself. However, applying the same methodology, other authors
confirmed that the serum metal increases were basically due to the rise of circulating
ceruloplasmin (59,82). As it was already reported in this paper, a
further and, perhaps, conclusive support for the latter evidence was obtained
directly quantifying the actual amounts of non-Cp-bound copper in the serum
of rheumatoid patients, in whom only minor variations of this circulating
fraction of the metal were observed (55). Finally, considering as a whole the
data summarized in Table 9, it appears that only very few studies reported
the total serum copper or ceruloplasmin concentrations as unchanged, and
none noted a decrease in the level of the above two parameters (3,76,77).
Total serum copper and ceruloplasmin do not change significantly in
relation to the dietary supply of this metal, unless a biologically significant
copper-deficient alimentary regimen is adopted, or relevant amounts of
intestinal copper-absorption inhibitors, such as zinc, iron, calcium, ascorbic
acid, phitate, etc., are present in the food (56,71,95). Conversely, the ceruloplasmin
concentration in the serum, and thus the total serum Cu, is strongly
influenced by a great number of physiological factors, (e.g., hormonal levels)
and pathological conditions (such as stress, inflammation, etc.) (17,56).
On the other hand, the erythrocytes are considered to be a blood compartment
in which copper is less susceptible to the hormonal as well as
pathological stimuli, unless the pathology considered is directly concerning
the erythrocyte itself (71,95). For this reason, the red blood cell copper level
is taken to be a relatively more reliable index of the actual overall body copper
status, at least as far as moderate deficiencies have to be identified (see
below for further details) (71,95). In general, the erythrocyte copper concentration
was found unchanged in this compartment of blood in RA
patients (Table 9), with the exception of two studies carried out by the same
research team that reported a significant increase of this parameter (59,86).
However, this last claim turned out to be a misleading result since, as
previously recalled in this paper, the apparent increase of the erythrocyte
copper level (measured as mg/mL of metal in the packed cells) was actually
due to the RA-induced significant decrease in the individual volume of red
blood cell, and the overall amount of copper contained in this total fraction
of blood [calculated according to the formula of Altman and Dittmer (70)]
resulted not to be significantly different from that of the age-matched
healthy controls (59).

182 Milanino
Another compartment that may be considered a somewhat good index
of a possible copper deficiency status is the 24-hour urine, albeit, as for the
total serum copper, the level of urinary copper is sensitive to biologically significant
body metal depletion, but not predictive of a marginal deficiency
condition (71,95). This index was found to be decreased only in one case
of acute inflammation [i.e., in the sandfly fever virus infection, carried out
on healthy volunteers (74)]. In the rheumatoid arthritis patients, an early
report of increased copper urinary excretion was published, whereas more
recent works found this parameter unchanged, thus convincingly suggesting
that RA does not promote a significant increase of body copper losses via
this excretory pathway (59,79,86,88).
To our knowledge, the copper status in an inflamed human solid tissue
was studied only in patients suffering from acute periodontal disease, in which
a 15-fold increase of the metal (measured as Cp levels) was found in the inflamed
as compared to the normal periodontal tissue explants (2). A few more
data exist on the copper content in the synovial effusions withdrawn from the
actively inflamed joints of RA patients, compared with the fluids taken from
the human osteoarthritic or traumatic knees used as control values
(75,76,80,84). In one single case, the copper concentration of the rheumatoid
synovial fluid was found to be insignificantly different from that measured in
the osteoarthritic subjects, whereas in the other instances a dramatic increase
of the metallo-element levels was measured in this particular compartment
(75,76,80,84). We would like to stress that this evidence, together with that
obtained evaluating the status of copper in human inflamed blood,
closely recalls the behavior of the metal metabolism previously described when
studying the development of acute and chronic inflammations in the
laboratory animal.
The data summarized in the above section of the paper, together with
other published information, may help in the attempt to propose some
reasonable answers to the questions with which this survey on the human’s
‘‘endogenous’’ copper metabolism in inflammation (especially RA) has been
introduced. The hypothesis that a marginal copper deficiency could contribute
to the development of the rheumatoid arthritis was detailed by
Rainsford and Sorenson (90–92). These authors reported that there was
evidence suggesting that the dietary intake of copper in some Western countries
might be well below that required for natural physiological functions.
Moreover, it was pointed out that high levels of environmental pollutants,
toxic metals such as lead and especially cadmium, might act as antagonists
of copper absorption or affect the normal development of its biological
pathways (91). However, although at least in the United States, the average
intakes of copper may even be below the FDA’s recommended dietary
allowances, there are still conflicting opinions about the practical need for
concern over health in relation to copper status in humans (96). Moreover,
this issue cannot be directly solved using the techniques available today for

The Role of Copper in Inflammation 183
defining the existence of a marginal copper deficiency. In fact, the serum
copper and Cp concentrations may well reveal a moderate or severe metal
deficiency but are not at all responsive to marginal deficiencies (97,98). The
same lack in the capacity to reveal a marginal copper-deficiency condition is
true for both the 24-hour-urine metal excretion and the total copper red blood
cell level (98). Conversely, the evaluation of a body copper marginal deficiency
could, perhaps, be achieved by studying erythrocyte SOD 1 activity, although
an early work reported that this parameter might increase in conditions in
which an oxidative stress of any origin has been caused (97,99,100). More
promising seems to be the measure of the cytochrome-c oxidase activity in
the platelets. Actually, studies with rats show that this enzymatic marker is
a sensitive sign of copper stores (101,102); the cytochrome-c oxidase
activity in platelets highly correlates with copper concentration in the
liver (r ¼ 0.99, P < 0.0001), an established indicator of copper status in
animals (97). In conclusion, the hypothesis that a marginal copper deficiency
status could favor the onset of the human rheumatoid arthritis cannot currently
be either confirmed or denied. On the contrary, according to data
available today, it seems reasonable to suggest that adequately nourished
RA subjects do not develop a significant body copper depletion over time
(59,85). In particular, studying the copper content of plasma, blood cells,
and 24-hour urine in rheumatoid arthritis patients divided into four groups
according to their disease duration, i.e., 0–1, 1–5, 5–10, and over 10 years,
we have clearly shown that all the above-mentioned parameters were not significantly
influenced by the duration of the disease itself (59). It seems worthwhile
to stress that, at least for the urinary copper excretion and copper
erythrocyte levels, a progressive decrease of these indices would have to be
observed if the disease were responsible for a time-dependent biologically significant
depletion of body copper stores (71,95). Consequently, it appears reasonable
to exclude the need for a dietary copper replenishing therapy in
response to the hypothesized RA-induced copper deficiency in humans suffering
from rheumatoid arthritis. Finally, focusing on the possibility that the total
serum (or plasma) copper could be taken as a predictive clinical marker in the
rheumatoid patient, this speculation has been belied by recent work (59,85). For
instance, plasma copper was found to correlate significantly with some other
plasma indices of RA, such as the iron concentration (r ¼ 0.20, P < 0.050), total
proteins (r ¼ 0.33, P < 0.010), a2-globulins (r ¼ 0.46, P < 0.010), c-globulins
(r ¼ 0.22, P < 0.050), and ceruloplasmin (r ¼ 0.77, P < 0.001), but no significant
correlations were found versus the well-established major clinical
markers of the disease, i.e., the number of swollen joints, the grip strength,
the functional class, the anatomical stage, and the physician’s assessment
index (59). Interestingly enough, the above data confirm, adding more
sophisticated details, the evidence already reported in this review when discussing
the acute and chronic animal models of inflammation, which led us
to propose that the increase of total serum (or plasma) copper is most

184 Milanino
likely an ‘‘all or nothing’’ phenomenon, unable to discriminate between
mild and severe inflammatory conditions.
EFFECTS OF ‘‘EXOGENOUS’’ COPPER ADMINISTRATION
ON THE INFLAMMATORY PROCESS
The data summarized so far may be briefly outlined as follows:
Dietary copper deficiency acts as a proinflammatory stimulus, at
least in animal models of acute inflammation.
In rats on a normal Cu-containing diet, the induction of acute and
chronic inflammatory processes clearly produces a condition in
which more copper is required by some compartments of the
organism. In view of the fact that no major redistribution of ‘‘endogenous’’
copper occurs, it seems reasonable to suggest that this
increased demand for the metal is mainly fulfilled by enhanced
intestinal absorption and/or decreased intestinal excretion of copper
(37,57). Interestingly, it has been shown that turpentine edema
increases the amount of dietary copper required to maintain hepatic
SOD 1 levels equal to those of nonstressed rats (103). Moreover,
albeit limited to blood, urine, acutely inflamed periodontal tissue,
and rheumatoid synovial fluids, the data reported in humans would
appear to give further support to the above hypothesis (57). Due to
reasons that are not yet understood, however, the natural antiinflammatory
effect promoted by increased intestinal absorption
and/or decreased intestinal excretion of the metallo-element appears
to be susceptible to significant improvement by the therapeutic
administration of copper compounds (7).
Thus, the use of ‘‘exogenous’’ copper preparations as remedies to counteract
the inflammatory pathologies appears to consistently find its rationale in
the above-summarized remarks. In turn, the study of anti-inflammatory potential
of administered copper would help to better understand the overall network
of relationships existing between this metal, both ‘‘endogenous’’ and ‘‘exogenous,’’
and the onset, development, and remission of the inflammatory process.
Problems Related to Routes of Copper Administration
The final therapeutic goal regarding the use of copper preparations as
anti-inflammatory agents is to identify effective and safe drugs to keep
human rheumatoid arthritis, an important deabilitating disease for which
a fully satisfactory treatment has not yet been found, under adequate control,
if not to cure it (104). To reach this goal, the physician should not only
consider an effective and safe copper compound, but also be able to administer
this potential drug (which could be possibly used for long periods of
time) by a nontraumatic route like oral or topical.

The Role of Copper in Inflammation 185
Many reviews have been published summarizing the anti-inflammatory
properties of over 140 different copper(II) complexes with a wide variety of
ligands (92,105–108). Examination of the literature reveals that for the
large majority of these copper-containing molecules the anti-inflammatory
and/or antiarthritic activity was evaluated in animal models, following sc
or intraperitoneal (ip) administration. In fact, sc and ip treatments are preferred
to demonstrate and reproduce in vivo biological activity of copper.
Copper(II) preparations given by any parenteral route tend to disappear
rapidly from plasma, being mainly accumulated in the liver and, to
some extent, in the kidneys (109). However, when these substances are
administered orally, they may not enter the organism unchanged, and their
copper content may not become bioavailable, unless very special Cu(II)
ligands or particular vehicles and adequately high amounts of complexes
are used. Most copper compounds, when exposed to the acidic pH in the
stomach, actually undergo nearly complete dissociation, followed by formation
and absorption of new, in situ formed Cu(II) complexes (110,111).
Copper is then transferred from the portal circulation to the liver, which
regulates its subsequent metabolism by:
Storing the metal as Cu-thionein in the hepatic tissue, thus creating
a physiological reservoir of the metal to be used according to
actual physiological needs (56).
Completing the synthesis of the copper-dependent proteins, a
process in which a membrane copper P-type ATPase, the Wilsondisease
protein (WND) (112), seems to be involved in delivering
Cu(I) ions into the Golgi’s vesicles. In particular, the WND protein
acts co-operatively with the ClC-4 chlorine channel (113) to assemble
the olo-ceruloplasmin in this subcellular apparatus before the
multifunctional protein is secreted into the general circulation.
Finally, excreting the excess copper via the bile, once again
utilizing the activity of the WND protein assisted by that of the
Cu-chaperon Murr 1 (114).
As previously stressed the Cp synthesis is not induced, during conditions
of adequate dietary copper intake, by increasing the amount of the
metal present in the diet, nor by supplementing the organism with copper
given by any parenteral route (17,56,62,109). On the other hand, the
homeostatic mechanisms regulating the Cp production and secretion appear
to react exclusively to endogenous stimulations (17,56). Therefore, if the
orally administered copper complex is broken down by gastric juice, the extra
copper that enters the liver will, most likely, undergo hepatic first-pass
clearance, becoming not bioavailable for any therapeutic purpose.
Theoretically, topical administration of copper is the most convenient
method of treating rheumatoid arthritis, because it may avoid or minimize
the risks of systemic toxicity, and it may direct the drug straight to the

186 Milanino
affected area, i.e., the inflamed joints. The main problem with such treatment
is to identify both ligands and formulations that allow to carry therapeutically
significant amounts of the active principle through the skin. This problem is
made worse by the poor lipophilic nature of most Cu(II) compounds. Thus,
the goal to get any important clinical success using topically applied copper
preparations in the treatment of RA patients has not yet been accomplished.
Last but not least, the iv route of administration has to be considered,
although in recent times it only has been used in a few cases. Nevertheless,
two of those deserve mention. In the first case, a number of copper-containing
proteins was tested for their anti-inflammatory potential, and found to be
active in an acute animal model of inflammation. In the second, and certainly
in a more significant one, the antirheumatic action of a copper(II) complex
with salicylic acid has been clinically evaluated in man. This latter study
appears to bear even nowadays great interest, both theoretical and practical.
We would like to open our survey on the effects of ‘‘exogenous’’ copper on the
development and control of inflammation, reporting on iv administration of
these metal-containing molecules.
Intravenous Administration of Copper Compounds
Laroche and coworkers (20) isolated a number of copper-dependent enzymes
from different animal and vegetable sources, and iv-tested them for their
acute anti-inflammatory activity in the laboratory animal, using yeastinduced
paw edema model in the mouse (Table 10).
As reported in the table, the copper proteins ceruloplasmin (isolated
from beef erythrocytes) and laccase [isolated from a fungus belonging to
the genus Polyporus (70)], which are both biochemically classified as ‘‘bluecopper
oxidases,’’ appear to be the more active among the tested molecules.
It is relevant to note that, as stressed by the authors to validate their results,
Table 10 Examples of Anti-inflammatory Activity of Some Cu-Dependent
Enzymes Administered iv in the Mouse, Challenged with a Subplantar Injection of
a Yeast Suspension
Copper-dependent enzyme Source Edema inhibition (%)
Cu-serum albumin Human serum None
Ceruloplasmin Beef serum 73
SOD 1 Beef erythrocytes 44
Diamine oxidase Pig kidney 35
Cytochrome oxidase Pseudomonas aeruginosa 53
Ascorbate oxidase Cucumis sativus 37
Laccase Polyporus versicolor 72
Abbreviations: SOD 1, cytoplasmatic Cu,Zn superoxide dismutase; iv, intravenous.
Source: From Ref. 20.

The Role of Copper in Inflammation 187
the anti-inflammatory activity was measured three hours after the challenge
to which the iv treatment followed immediately. This makes it possible to
use heterologous proteins without inducing any immune reaction in the
experimental animal (20).
As mentioned regarding intravenous use of copper complexes in
humans, a 20-year-long clinical study demands attention. Hangarter (115)
reported that in the treatment of arthritic diseases the therapeutic results
with iv copper alone were comparable to those of chrysotherapy, although
copper treatment did not give rise to the considerable side effects associated
with gold-salts treatment. However, Hangater’s major efforts were dedicated
to clinical evaluation of a copper complex marketed as Permalon TM .
Chemically, the antirheumatic drug Permalon is a complex of one or two
cupric ions coordinated by two or four salicylate molecules, i.e., Cu(II)-
(salicylate)2 or Cu(II)2-(salicylate)4 (105,116). In the period between 1950
and 1971, Hangarter (115,116) studied the activity of Permalon in 1147
patients suffering from different rheumatic-degenerative diseases such as the
arthrosis deformans, sciatica, erythema nodosum, lumbar spine syndrome,
cervical spine–shoulder syndrome, acute rheumatic fever, and, particularly,
rheumatoid arthritis. Focusing on the RA patients, the treatments with slow
iv infusions of cupric salicylate followed the protocol reported in Table 11,
where the clinical results obtained are also summarized.
Commenting on the above results the author stated that, according
to own experience, the antirheumatic effects obtained with intravenous
cupric salicylate were considerably superior to those achieved with the
antirheumatic agents commonly used at that time, such as nonsteroidal
anti-inflammatory drugs (NSAIDs), antimalarial agents, gold salts, as well
as cortisone preparations (116). Interestingly, during the open discussion
that followed the presentation of that paper at an international symposium
held in Little Rock (Arkansas) in 1981, it emerged that the RA patients
who responded to the therapy with remission of the disease maintained
Table 11 Protocol of Permalon Treatment Within 620 Rheumatoid Arthritis
Patients, and Results Obtained by the Therapy—A 20-Year Study
Treatment 6–10 infusions at intervals of 2–4 days
Composition of each infusion Salicylate 6–8 g; copper 7.5–10.0 mg
Salicylate: maximum dose for a full cycle 80 g, i.e., average of 2.5 g/die
Copper: maximum dose for a full cycle 100 mg, i.e., average of 3.1 mg/die
Therapeutic effect reported:
Remissions 403 cases (65%)
Significant improvements 143 cases (23%)
No effects 74 cases (12%)
Source: From Ref. 116.

188 Milanino
their completely symptom-free status for a subsequent average period of
three years (116). The therapy was occasionally accompanied by a transient
nausea and tinnitus, both of which are very well-known minor side effects
due to the administration of high salicylate doses (117). However, no other
adverse reactions were observed during the treatments as well as the subsequent
follow-up. In particular: (i) no disturbances were reported at the
level of the gastrointestinal tract; (ii) the blood sugar, electrolyte levels, and
the classical serum electrophoresis markers were maintained within the
normal ranges; and finally, (iii) no evidence of cardiac, circulatory, renal, hepatic,
respiratory, or central nervous system toxicities were found (115,116).
Unfortunately, the above-reported results were obtained following the criteria
of an open trial (i.e., in the absence of a double-blind comparison versus both
sodium salicylate- and placebo-treated control groups); consequently, they
cannot be taken as a valid report on the basis of clinical protocols of evaluation
accepted today. In 1971, Prof. Hangarter retired; the production of
Permalon was discontinued by the manufacturer for economic reasons
(116), and since then no other research team decided to submit cupric salicylate
to a currently officially approved clinical trial.
Subcutaneous and Intraperitoneal Administration
of Copper Compounds
Table 12 shows a few representative examples of copper compounds, such as
simple copper salts, copper-containing proteins, and copper complexes with
widely different inorganic and organic ligands, which have been shown to
possess significant anti-inflammatory and/or antiarthritic properties following
sc or ip injections in the experimental animals. Notably, a number of
these copper complexes are formed using NSAIDs as complexing agents.
Although today well established at least in the animal models, the
anti-inflammatory activity of copper compounds subcutaneously or intraperitoneally
injected, but also given orally, has been the subject of a dispute
originated by the actual irritating potential that copper ions could exert at
the site of exposure (110,129). In the classical models of experimental acute
inflammation, the compounds to be tested are administered one hour before
the inflammatory challenge, and their activity is evaluated between three
and seven hours after the challenge itself (68,69). Bonta and Noordhoek
(130) showed that a preventive ip injection with a well-known promoter
of acute inflammation, i.e., a carrageenan suspension, was able to strongly
inhibit the normal course of the subsequent inflammatory reaction induced
by subplantar injection of a kaolin preparation. The authors proposed
that the challenge with an irritant (or potentially irritant) substance done
at one site of the body could trigger the organism’s physiological antiinflammatory
response, thus enabling the living system to reduce the degree
of the inflammation later on induced by the same, or any other irritant,

The Role of Copper in Inflammation 189
Table 12 Representative Examples of Copper Compounds Active as In Vivo
Anti-inflammatory and/or Antiarthritic Agents, Following sc or ip Administration
in the Animal (Rodents) Models of Inflammation
Compound Administration route Reference
Cu(II)Cl2 sc 118
Cu(I)2O sc 119
Cu(II) 2-(acetate) 4 sc 7
Cu(OH)2CuCO3 [basic cupric
sc 120
carbonate (malachite)]
Cu(I)-thiomalate sc 121
Cu(II)-ascorbate sc 7
Cu(II)-(anthranilate)2 sc 7
Cu(II)2-(3,5-DIPS)4 sc 122
Cu(II)-diaqutetrakis(p-cresotate)-(H2O) 2 ip 123
Cu(II)-diaqutetrakis(o-cresotate)-(H2O) ip 124
Cu(II)-histidine ip 125
Cu(II)-tryptophan ip 125
Cu(II)-cysteine ip 125
Cu,Zn-SOD (from bovine liver) sc 126
Cu(I)-thionein ip 127
Cu(II)2-(salicylate)4 a
sc 122
Cu(II)2-(aspirinate)4 a
sc 122
Cu(II)n-(niflumate)2n-(H2O)n a
sc 122
a
Cu(I)-D-penicillamine-(H2O) 1.5 sc 122
Cu(II)n-(fenamole)n-(acetate)2n a
sc 122
Cu(II)-piroxicam a
ip 128
a
The anti-inflammatory activity of the copper complex is significantly greater than that of the
parent ligand used in the experiment.
Abbreviations: sc, subcutaneous; ip, intraperitoneal.
injected at a remote site of the same organism; this phenomenon was labeled
‘‘counter-irritancy’’ (130). Thus, the parenteral and oral irritation usually
ascribed to copper suggested that the anti-inflammatory activity seen with
the copper compounds could be explained on the basis of their ability to
promote a ‘‘counter-irritant’’ reaction (129,131). However, this hypothesis
was consistently disputed by other published data, according to which the
‘‘counter-irritancy’’ of copper compounds was only negligibly responsible
for their anti-inflammatory activity (7,132,133). Recently, we carried out
an experiment in the attempt to better clear up this issue (the paper has been
submitted for publication). Noninflamed female rats were subcutaneously
injected with aseptic isotonic solutions of Cu(II)-(acetate)2 containing
either 3.0 [i.e., a fully therapeutic dose (7,105)] or 0.3 mg/kg of copper.
The animals were sacrificed three, seven, or 24 hours after treatment. Skin
specimens of the injected area were removed to perform a full macroscopic

190 Milanino
and microscopic histological examination, and the blood was collected to
determine its plasma total copper concentration. The results obtained
unequivocally pointed out that, at all considered times, the histology did
not showed any significant sign of a local inflammatory response. Moreover,
the plasma total copper remained unchanged throughout the experiment,
compared with that measured in the saline-treated controls. In particular,
this significant marker of inflammation did not increase 24 hours after
the sc injection of either copper acetate solutions. The latter evidence seems
to convincingly suggest that the treatments did not induce a systemic antiinflammatory
reaction capable of interfering with the own anti-inflammatory
potential of copper, further sustaining that the pharmacological action of
the metal is real, and that it does not depend on any biologically relevant
‘‘counter-irritancy’’ phenomenon.
As mentioned in the introduction, Sorenson (7), in his classical paper,
not only showed that many sc given copper-containing molecules are very
active anti-inflammatory and/or antiarthritic agents, but also proposed a
theory that was destined to open an active debate. Sorenson’s (92,107) idea
was that the coordination compounds formed in vivo between the surplus
of copper present in the plasma of inflamed animals or humans and the clinically
used nonsteroidal anti-inflammatory agents could represent the active
metabolites of these drugs, and moreover, Sorenson suggested that
the superoxide radical disproportionation (i.e., SOD 1 mimetic activity)
accounted for the anti-inflammatory action of these copper(II) complexes.
Sorenson’s hypothesis was, however, challenged by coordination chemists
who, mainly, if not exclusively, using a computer simulation approach,
claimed that the complexes between copper(II) and salicylate, acetylsalicylate,
as well as many other NSAID-type counter-anions, could not exist
under plasma conditions since the ligands are incapable of effectively competing
for the metal (108,134–136). Nevertheless, the original observations
made over the years that copper chelates of anti-inflammatory drugs have
greater potency as anti-inflammatory agents can still be demonstrated with
a considerable number of compounds (137). In particular, in a variety of
laboratory animal models, parenterally administered copper aspirinate
showed to posses relevant anti-inflammatory activity beyond that provided
by an equimolar amount of aspirin (137). Referring to aspirin, it seems appropriate
to recall that oil–water partition coefficient studies have shown
that copper aspirinate, likely in the form Cu(II)2(aspirinate)4, is 10 times
more lipophylic than aspirin at comparable pH values of about 4.0 (108,138).
Obviously, this implies that copper aspirinate, once entered into the organism,
is facilitated in diffusing through the lipophilic cellular barriers and,
thus, more bioavailable than aspirin. Another interesting observation was
the result of the studies carried out with a compound closely related to the
salicylate, i.e., the 3,5-di-isopropylsalicylate (3,5-DIPS), which, although
has no anti-inflammatory activity as such, was shown to possess potent

The Role of Copper in Inflammation 191
anti-inflammatory activity when complexed with Cu(II) ions (105). It was
found that in plasma Cu(II)(3,5-DIPS)2/Cu(II)2(3,5-DIPS)4 forms stable
complexes with human serum albumin (139,140). This, in turn, led researchers
to suppose that other, either known or unknown endogenous copper
ligands, could allow the existence of some Cu(II)-NSAID or non-NSAID complexes
in vivo, by coordinating them in sort of a multimolecular adduct able to
survive as such in plasma or in interstitial fluids. Finally, to further sustain the
soundness of the Sorenson’s hypothesis, two other in vivo studies deserve to be
mentioned. In the first, Korolkiewicz and coworkers (141) showed that copper
aspirinate is significantly more active than an equimolar mixture of copper and
aspirin, when given orally to inflamed rats. This observation seems to indicate
that, in spite of any theoretical prevision obtained from computer simulation
models, some copper aspirinate complex should survive intact at the acid pH
of the stomach, and be absorbed by the animals treated. In the second study,
Milanino and coworkers (142) demonstrated that a standard oral dose of
indomethacin (i.e., 2 mg/kg) was remarkably active in reducing the carrageenan-induced
rat paw swelling (average inhibition: 49%). However, the drug lost
about 60% of its potency when administered to inflamed copper-deficient rats.
Oral Administration of Copper Compounds
As noted before, the oral route of administration of copper compounds
appears to be inconvenient, in general it being improbable that these molecules
would survive as intact Cu-complexes in gastric juice. Nevertheless,
there are examples that show that this problem could be overcome (Table 13).
Table 13 Some Representative Examples of Copper Compounds Active as In Vivo
Anti-inflammatory and/or Antiarthritic Agents, Following Oral Administration in
the Animal (Rodents) Models of Inflammation
Compound Vehicle Reference
Cu(I) 2O Mulgofen a
131
Cu(II)-(acetate)2 Isotonic solution 143
Cu(II)-(diaminoethane)2-Cl2O Mulgofen a
144
Cu(II)-D-alanine Mulgofen a
144
Cu(II)2-(3,5-diisopropylsalicylate)4 Propylene glycol 92
Cu(II)-bis(2-benzimidazolyl)thioethers Gum Arabic 145
b
Cu(II) 2-(aspirinate) 4 Starch mucilage 141
Cu(II)2-(aspirinate)4 b
Sunflower oil 146
Cu(II)2-(benoxaprofen)2 b
Sunflower oil 146
Cu(II)-carbonate Diet 62
a
Polyoxyethylated vegetable oil.
b
The anti-inflammatory activity of the copper complex is significantly greater than that of the
parent ligand used in the experiment.

192 Milanino
One possibility is to use as ligand a structure able to form stable complexes
with Cu(II) ions, thus obtaining a significant degree of resistance to
acid attack in the stomach. As a matter of fact, that is the case for the
unsubstituted bis(2-benzimidazolyl)thioether (NSN), which is able to bind
Cu(II), forming (both in the solid state and in solution) an adduct in which
the metal is penta-coordinated by the two benzimidazole N donors and the
thioether moiety, all belonging to the ligand, as well as by one water molecule
and one perchlorate group (145,147). Significantly, the Cu(II)-NSN
is stable enough in vitro to be intact at over 90% of its original amount,
at a pH ranging between 4.5 and 4.0 (145). Moreover, about 10% of the
complex is still present as such, well above the in vitro addition of the stoichiometric
quantities of HCl (i.e., two equivalents) required for the complete
protonation of the ligand (pH values near 3.0) (145,147). Thus, as can be
reasonably expected to be seen in the above evidence and the intrinsic
anti-inflammatory potential of copper, the Cu(II)-NSN complex was shown
to be orally active in both the acute (carrageenan paw edema; maximum
inhibition 57%, P < 0.001) and chronic (adjuvant-induced arthritis; maximum
inhibition 46%, P < 0.01) models of rat inflammation, whereas NSN
alone was virtually devoid of any anti-inflammatory activity (145). Moreover,
in the acute model the pharmacological response of Cu(II)-NSN
showed to be clearly dose-dependent, thus validating the biological meaning
of the anti-inflammatory (and antiarthritic) effect observed (145). Unfortunately,
it was not possible to carry out further studies on this interesting
molecule. Consequently, it remains unknown whether in vivo it acts as
intact Cu(II)-NSN or it expresses its pharmacological activity upon transformation,
by the organism’s metabolism, into a chemically different active
molecule. Moreover, there is no information about its possible mechanism(s)
of action as an anti-inflammatory agent, in vivo or in vitro. Conversely,
another potentially strong copper-complexing molecule, i.e., the D-penicillamine
(a drug still commonly used for the treatment of Wilson’s disease) (9), has been
given orally, as Cu(II)-D-penicillamine complex to rats affected by the kaolininduced
paw edema, but it remained inactive also at the very high dose of
300 mg/kg (144).
An alternative way to overcome the gastric barrier action is to use vehicles
able to protect the integrity of the orally administered copper complex,
thus assuring both its absorption and bioavailability. One such a vehicle
appears to be sunflower oil. In fact, suspending the Cu(II)2(aspirinate)4 complex
in sunflower oil, Rainsford (146) was able to show that 150 mg/kg of
orally administered complex caused a 38% inhibition (P < 0.05) of the rat
paw swelling (carrageenan-induced inflammation), whereas the same dose
of aspirin alone was totally inactive. Similar results were also obtained studying
the copper complex with another NSAID, i.e., the benoxaprofen (146).
Thus, 100 mg/kg of Cu(II)2(benoxaprofen)4 exhibited a 53% inhibition of
the edema, whereas 100 mg/kg of the ligand alone were fully inactive.

The Role of Copper in Inflammation 193
Interestingly, both NSAID-copper complexes not only showed a significantly
greater potency compared with the parent drugs, but they were also virtually
devoid of the gastric ulcerogenic activity that characterized the benoxaprofen
(upon repeated daily oral dosing), and in particular aspirin (146). Evidence
that Cu-NSAIDs complexes have a much lower gastric toxicity than the
parent compounds has been first reported and then repeatedly confirmed
by Sorenson (92,105,107,122). Another vehicle that could favor the absorption
of intact copper complexes following their oral administration is a
polyoxyethylated vegetable oil named mulgofen. For instance, some copper
complexes, such as the Cu(II)-(diaminoethane)2 and Cu(II)-D-alanine,
resulted to be active inhibitors of the kaolin-induced rat paw edema model
when suspend in mulgofen and given orally at 100 mg/kg (144). However,
using the same vehicle, oral aspirin and copper aspirinate were both found
to be inactive at the dose of 100 mg/kg (131). Nevertheless, regardless of
the vehicle used, it seems reasonable to point out that a significant
number of studies, which have been reviewed recently (92,148), showed that
a remarkable number of copper complexes, and those formed using many
NSAIDs as ligands, in particular, are active anti-inflammatory and/or antiarthritic
agents not only after iv, sc, and ip dosing but also following oral
administration, provided that sufficiently high amounts of the compounds
are given.
Since it was not possible to show a proinflammatory effect of dietary-induced
copper deficiency on the adjuvant-arthritic rat (26,27,33), the
behavior of the metal in this experimental chronic pathology was studied
by carrying out an ‘‘opposite’’ experiment, i.e., examining the development
of the AA in rats fed a copper-supplemented diet. Using this simple ‘‘dietary
trick,’’ it was possible to examine the effects of the oral copper administration
on the inflammatory process, excluding, at the same time, any possible
biological actions of the ‘‘exogenous’’ ligands used to form the conventional
copper complexes commonly studied as anti-inflammatory/antiarthritic
drugs. According to a preliminary observation, a putative anti-inflammatory
diet containing 200 ppm of copper (added as carbonate; control diet,
Cu ¼5 ppm) was actually able to inhibit the development of the adjuvant
arthritis ( 28%, P < 0.01) in the copper-supplemented rats after one month
of ‘‘prophylactic’’ feeding (149). Thus, the approach of studying the pharmacotoxicology
of the 200-ppm copper-supplemented animals was continued,
evaluating the development of the AA in rats preliminarily treated with
experimental diets containing 50, 100, or 200 ppm of copper (i.e., respectively,
10, 20, and 40 times the standard copper requirements) (62,63,150).
The results obtained from the toxicological analyses on noninflamed rats
after 30 and 58 days of 200 ppm copper-supplemented feeding are reported
in Figure 3 (relative to the changes of copper status in the plasma, liver,
and kidneys) and in Table 14. The data plotted in Figure 3 show that the
200 ppm Cu-supplemented diet was able to progressively and significantly

194 Milanino
Figure 3 Status of copper in plasma, liver, and kidney of noninflamed female rats
following 30 and 58 days of feeding with either a normal (Cu ¼ 5 ppm) or supplemented
(Cu ¼ 200 ppm) copper diet. On the right side of each column referring to the
dietary ‘‘copper-supplemented’’ rats (Cu ¼ 200 ppm), the percent increases of copper
versus the time-matched control diet (Cu ¼ 5 ppm) are reported in absolute values.
Statistics (Student’s t test): P < 0.01. Source: From Refs. 62, 63, and 150.
increase the total copper content in the liver and in the kidneys above the control
values, following 30 and 58 days of treatment. Conversely, as expected and
repeatedly stated above, such pronounced copper supplementation did not at
all influence the metal status in the plasma of the animals. It is worth stressing
that, although the same trend described above was observed in the 50 and
100 ppm Cu-supplemented rats, the increases in hepatic- and renal-copper
measured were remarkably lower compared to those obtained with the
200 ppm copper-treated animals, and throughout the experiment were not
significantly different from the control values (61). Table 14 shows that the
200 ppm copper-containing diet notably increased the total amount of
copper present in the hind paws also, albeit this effect was remarkably more
evident at day 30 than at day 58.
However, possibly more interesting are the following evidences:
Copper supplementation did not modify the status of the metal in
the cell fraction of blood, as well as in the brain of the treated rats.
Massive dietary copper supplementation did not change the zinc
status in any of the compartments examined. This observation is
very relevant since, on the one hand, the excess of alimentary

The Role of Copper in Inflammation 195
Table 14 The Toxicology of the Non-inflamed Copper-Supplemented Rats
Parameter
Changes versus the
normally fed rats
(Cu ¼ 5 ppm)
30 days 58 days
Hind paw copper þ36% a
þ17% a
Blood cells and brain copper None None
Plasma, blood cells, liver, kidney, brain,
and hind paws zinc
None None
Hematocrit None þ3% b
White cells (count, differential count) None None
Red cells (count, mean erythrocyte volume) None None
Platelets (count, mean platelet volume) None None
Serum aspartate aminotransferase None None
Serum alanine aminotransferase 16% b
None
Serum alkaline phosphatase None 33% b
Serum creatinine None None
Serum urea None None
Hemoglobin 2% b
None
Sodium, potassium, chlorine, calcium, and CO2 None None
Blood proteins (total and fractionated, albumin,
a/c ratio)
None None
Macroscopic and microscopic examination of liver,
kidneys, adrenals, hind paws, eyes, thymus,
lymph nodes (cervical, mesenteric), salivary glands,
esophagus, stomach, duodenum, jejunum, ileum,
cecum, colon, rectum, spleen, pancreas, trachea,
lungs, heart, aorta, skin, mammary glands,
urinary bladder, prostate, ovary, thyroid,
parathyroids, brain, pituitary, spinal cord,
sciatic nerve, skeletal muscle, femur, and sternum
None None
Note: A summary of the parameters (other than those reported in Fig. 3, i.e., the plasma, liver,
and kidney copper values) evaluated after 30 and 58 days of feeding with the ‘‘anti-inflammatory’’
200 ppm copper-containing diet.
a P < 0.01.
b P < 0.05 (Students’ t test).
Source: From Refs. 62 and 150.
copper might induce an inhibition of the intestinal zinc absorption,
and, on the other, zinc has been shown to be an ‘‘endogenous’’ as
well as an ‘‘exogenous’’ anti-inflammatory agent (56,151).
The parameters related to the main toxicological markers of the
blood cell, blood proteins, and hematochemical conditions,
revealed that they were not affected by the treatment.

196 Milanino
The major indices of the hepatic and renal functions were found
to be within normal ranges. This evidence bears particular significance,
since both liver and kidney (together with the brain and, to
some extent, the erythrocytes and bones), are well-known target
organs for copper toxicity (9,152).
Finally, the macro- and microscopic examination showed that all tissues
listed in Table 14 have fully normal anatomical characteristics.
As far as the antiarthritic activity is concerned, it should be pointed
out that the tail injection of the complete adjuvant was done after 30 days
of preliminary feeding with either the control (Cu ¼ 5 ppm) or any of the
three copper-supplemented diets. The development of the chronic pathology
was then followed for 28 days, during which the dietary regimen of each
of the four groups of animals was kept unchanged (62,63,149). The results
shown in Figure 4 indicate that the 50 and the 100 ppm copper-containing
diets were not able to reduce the arthritic score at any considered time
point (i.e., 14, 21, and 28 days after the inoculum). Conversely, the 200 ppm
copper-supplemented diet, although ineffective at day 14 and 21, showed
a noteworthy and biologically significant inhibition of the arthritic score
% inhibition
of arthritic score
35
28
21
14
7
0
Cu = 200 ppm
Cu = 100 ppm
Cu = 50 ppm
14 21 28
Days after complete-adjuvant tail injection
Figure 4 Effects of three different copper-supplemented diets on the development
of adjuvant-induced arthritis in the rat. The plotted data represent the percent inhibition
of adjuvant-arthritis scores measured in arthritic rats fed with diets containing
10, 20, or 40 times the amount of dietary copper (Cu ¼ 5 ppm) present in the standard
diet given to the adjuvant-arthritic control p < 0.001. Source: From Ref. 62.
*

The Role of Copper in Inflammation 197
measured at the end of the experiment (day 28). The pharmacological importance
of the above results was confirmed when evaluating the total plasma
copper concentration in the arthritic rats fed the control diet versus those
kept on the 200 ppm copper-added one during the symptomatic phase of
the experimental disease (day 14 and day 28). Figure 5 shows that, different
from the AA-controls, the total plasma copper concentration in the 28-daysarthritic
copper-supplemented animals was dramatically reduced, which, in
turn, suggests that an amelioration of the overall clinical status of affected
rats may have occurred. The above speculation is further sustained when
% copper
increase
% copper
increase
150
120
90
60
30
0
150
120
90
60
30
0
AA day 14
plasma
AA day 28
*
hind paws
plasma hind paws
AA rats on copper normal diet AA rats on copper 40 times supplemented diet
Figure 5 Percent increases of total copper content in the plasma and hind paws of
adjuvant-arthritic rats, fed with either a standard (Cu ¼ 5 ppm) or copper-supplemented
(Cu ¼ 200 ppm) diet. Each column represents the percent increase in total
copper induced by chronic disease in affected rats, compared with their time- and
diet-matched controls, at 14 (upper panel) or28(lower panel) days after challenge.
Statistics (Student’s t test): all the values are statistically significant versus the respective
control groups; however, emphasis has been put on the comparison between total
plasma copper of arthritic copper-supplemented rats at day 14 and 28, P < 0.0001.
Source: From Ref. 63.

198 Milanino
taking into account other significant markers of the seriousness of the
experimental disease (Table 15).
For a correct understanding of the data reported in Table 15, it should
be recalled that the paw weight and the circulating white blood-cell and
blood-platelet numbers significantly increase in the AA animals; conversely,
a remarkable decrease of total kidney-copper amount and total plasma-zinc
concentration are brought about by the pathology, being inversely correlated
to its gravity (5,48,57). It thus becomes evident that the tendency
towards normalization shown by all the above-mentioned disease-induced
metabolic and physical changes (the arthritic score and the total plasma
copper concentration included) seems to testify in favor of the efficacy of
the 200 ppm copper supplementation in counteracting the development
of rat adjuvant arthritis. Finally, differently from plasma copper, the total
amount of the metal contained in the hind paws of the arthritic animals
(expressed as percent increases), does not seem to show biologically significant
changes comparing the data of day 14 with those of day 28, in both
copper-normal and -supplemented rats (Fig. 5). The maintenance of high
level of copper in the inflamed area might be simply due to a slow clearance
rate of the metal from this body compartment. However, considering that
28 days after the complete-adjuvant injection the experimental pathology
remains unresolved, it could be more likely that copper is kept within the
affected site in order to favor the local processes of tissue repair. This
hypothesis is supported by the evidence that the physiological equilibrium
of connective tissue depends upon a correct functioning of the copperdependent
enzyme lysyl oxidase (153–156). Moreover, a tripeptide-copper
complex, i.e., the glycyl-L-histidyl-L-lysine-Cu(II) (GHK-Cu), which is
naturally formed within the damaged tissue, appears to be also intimately
involved in tissue regeneration and remodelling process (155,157–160).
Table 15 Percent Differences of Some Markers of Adjuvant-Arthritis Severity in
Normally Fed and 200 ppm Cu-Supplemented AA Rats
Parameter
AA in the 5 ppm
copper-fed rats (%)
AA in the 200 ppm
copper-fed rats (%)
Mean weight of hinds paws (g) þ58 þ30 a
Kidney copper (total mg) 53 34 a
Total plasma zinc (mg/mL) 47 16 a
White blood cells (10 3 /mm 3 ) þ41 þ12 a
Blood platelets (10 3 /mm 3 ) þ46 þ13 a
Note: The values were measured at the end of the experiments (day 28).
Total number of rats: 30–40 per group.
a
P < 0.001 (Student’s t test).
Source: From Refs. 62 and 63.

The Role of Copper in Inflammation 199
Percutaneous Administration of Copper Compounds
Reports related to the concerns and merits of copper preparations topically
applied as anti-inflammatory/antiarthritic agents are thoroughly discussed
in other chapters of this book. Nevertheless, the issue of the treatment of
systemic inflammatory pathologies with percutaneous copper merits concise
mention also in this review. The efficacy of the cutaneous treatment with
copper-containing preparations in the therapy of wounds and some skin diseases
has been empirically known for more than 3500 years ago [papyri of
Ebers and Smith, circa 1550–1450 B.C. (6)]. Nowadays, the fact that tissue
repair requires the local presence of copper-dependent molecules is an
experimentally well-established evidence (155). The copper-dependent
enzyme lysyl oxidase belongs to these molecules, which catalyses the formation
of the cross-linking compounds that bind together the peptide chain of
collagen and elastin, and in so doing impart both support and elastic properties
to the tissues (153). Furthermore, when the extracellular-matrix macromolecules
are degraded by different pathological stimuli, the protein
cleavage leads to a virtuous feedback mechanism, yielding a number of peptides,
some of which act as potent signals for the repair and remodelling of
the damaged tissue (161). In particular, one of these peptides, the GHK-Cu,
appears to be extremely active in promoting the healing of excision wounds
and the skin damages caused by scald-burns (both experimentally induced),
probably by means of a multifaceted way of action (155,158). Notably, the
Cu(II)-tripeptide mentioned above is also fully active following topical treatments
(159). At least theoretically, it appears to be easy to obtain relevant
therapeutic results giving selected copper-containing preparations by cutaneous
route, in the case of open wounds and, perhaps, skin pathologies.
However, it is much more difficult to obtain similar effects using the same
route of administration on intact skin, as when one has to deal with chronic
systemic pathologies, such as adjuvant-arthritis of the rat and the human
rheumatoid arthritis. As reported above, in both chronic diseases the untreated
organism tends to accumulate copper within the inflamed tissues and
inflammatory exudates (48,75,80,84). According to the most currently accepted
interpretation, this particular event, together with the increases of the
total copper measured in some other body compartments, is likely devoted
to contrasting and eventually defeating the chronic pathology (151). This
hypothesis appears to be also supported by the observation that the concentrations
of copper in the paws of the 28-days-arthritic rats is increased by about
50% to 60% in both copper-normal and copper-supplemented animals, compared
to their respective time-matched controls (62,63). However, only in the
copper-supplemented arthritic rat, in which the absolute amounts of the metal
contained in the hinds paws is about 1.5 times greater than that measured in
the arthritic controls, a significant therapeutic effect was observed at the end
of the experiment (62,63). Thus, achieving an increased copper concentration

200 Milanino
within the inflamed site seems to be an important therapeutic goal using any
potential anti-inflammatory and/or antiarthritic copper-containing molecule.
The attempts at using percutaneous administration of copper in the
treatment of inflammatory conditions began with the studies of Walker
and coworkers (162,163), who claimed that wearing the copper bracelet
could have some beneficial effects on rheumatoid arthritis patients, and also
showed that the metallic copper might be actually dissolved by some sweat
components and permeate the intact cat skin. However, the research on the
anti-inflammatory potential of topically applied copper preparations was
quickly reoriented toward the use of nonphysiological cupriphores, as soon
as it became evident that nonlipophilic NSAIDs can form much more lipophilic
molecules by complexing with copper, and at the same time, they
proved to be more potent anti-inflammatory/antiarthritic agents compared
to the parent compounds (7,107,108,138). A number of studies have been
carried out using salicylic acid as cupriphore, although according to the
authors, at least one other NSAID, phenylbutazone, has also been, successfully
tested as a Cu(II) complex following percutaneous administration in
inflamed rats and horses (164,165). Focusing on Cu(II) salicylate, an ethanolic
preparation of this complex, registered as Alcusal 1 , has been the
subject of active research, done both in the laboratory animals and humans.
For instance, it was reported that topically applied Alcusal caused: (i) a significant
relief of swelling and joint stiffness in patients with rheumatoid
arthritis; (ii) suppression of established polyarthritis in rats; (iii) prevention
of the inflammatory response to the injection into the rat hind paw of carrageenan,
histamine, or hydroxyl apatite; (iv) minimal toxicity of the applied
compound in treated rats (166,167). Similar anti-inflammatory results were
obtained in laboratory animals, with Dermcusal 1 , a Cu(II) salicylate complex
in dimethyl sulfoxide/glycerol, and the choice of this vehicle was
claimed to favor the cutaneous absorption of the copper salicylate in comparison
with its parent, ethanol-based preparation (Alcusal) (168). Further
studies were carried out dissolving 64 Cu(II)-labeled salicylate in ethanol/
dimethyl sulfoxide/glycerol (66). This preparation was topically applied to
rats affected by two substantially different models of inflammation, i.e.,
the sponge granuloma (of about six days duration and of proliferative character),
and the carrageenan-induced foot edema (which lasts for a much
shorter time, and is mostly edemic in nature) (69). This kind of copper salicylate
preparations being already known for its anti-inflammatory activity
(167–169), the authors focused attention on the body labeled copper distribution
in the two models of inflammation. The data reported showed an
increase of radioactivity in the kidneys, liver, spleen, adrenals, thymus,
and serum from animals with long-lasting granulomatous inflammation. On
the other hand, no biologically significant percent changes of 64 Cu-relative
specific activity were observed in the tissues of rats with carrageenaninduced
paw edema (66). Thus, especially in the case of the rats affected

The Role of Copper in Inflammation 201
by sponge granuloma, it appeared that copper was actually absorbed from
the skin and then distributed systemically. Although promising on the basis
of the studies preformed with experimental animals, the anti-inflammatory/
antiarthritic potential of the above copper salicylate preparations topically
applied actually failed to be confirmed in humans. Their beneficial clinical
activity in osteoarthritic patients and in subjects affected by sports injuries
has been recently reviewed by Beveridge (164). The author based his claim
quoting only two unpublished studies with the Alcusal gel, which were, moreover,
carried out on a very limited number of patients (i.e., 20 and 19,
respectively). Conversely, a recent randomized, double-blind, placebo-controlled
trial done with topically applied copper salicylate gel on 93
patients with osteoarthritis of the hip and knee (170) was unsuccessful in
showing significant differences of the copper salicylate treatment compared
with controls that received placebo. Furthermore, significantly more patients
in the copper salicylate group reported adverse reactions, particularly
skin rashes (17.0% vs. 1.7% of the placebo group), which caused the exclusion
of these subjects from the trial (170).
Nonetheless, the contradictory data so far reported on the percutaneous
efficacy of copper in the therapy of human inflammatory conditions
should, by no means, discourage the pursuit of studies aimed at finding a
copper preparation able to efficiently penetrate the cutaneous barrier, and
to show a significant anti-inflammatory and/or antiarthritic activity in
humans. A recent study, in particular, continues in this effort. In fact,
Hostynek et al. (171) examined, by sequential tape stripping, the diffusion
of metallic pulverized copper through the human stratum corneum of intact
skin in vivo. Copper content was determined (inductively coupled plasmamass
spectroscopy) in 20 sequential strips taken from treated and control
subjects, either under occlusive (air exclusion) or semiocclusive (access of
air) copper applications, for periods of up to 72 hours. Focusing on 72-hour
exposure, and there on strips from no. 15 to 20 (that reach the glistening
epidermal layer, i.e., the vital layers of epidermis in intimate contact with
the derma postcapillary vascular bed), the following evidence was obtained:
(i) occlusive copper application let copper permeate the epidermis to a limited
extent only; (ii) conversely, under semiocclusive conditions, the presence
of both oxygen and some unidentified molecules contained in skin exudates
oxidizes Cu(0), and the in situ formed complexes entered the skin accumulating
within the stratum corneum in biologically significant amounts; and
(iii) in particular, at 72 hours, an average of about 0.6 mg/cm 2 of copper
was measured in each of layers from 15 to 20. Thus, assuming application
of copper on a surface of circa 100 cm 2 (which is a reasonable area to be
topically treated in the case of an inflamed knee joint), 72 hours after a single
treatment the total amount of metal that will accumulate in the inner
layer of epidermis (strip no. 20) will be equivalent to 60 mg of copper. Actual
bioavailability of such a relevant quantity of the metallo-element is

202 Milanino
unknown; however the evidence stated is a first, coming from direct measurements
showing potential, biologically significant concentrations of ionic
copper reaching selected anatomical locations in the body following percutaneous
treatment. Although in all likelihood most of the metal that
permeated the superficial skin layers under such experimental conditions
will be lost due to the effect of epidermal desquamation, since it is complexed
with molecules that are per se subject to the natural process of
excretion, the above results underscore the possibility of successfully using
the topical route of administration for copper.
In our opinion, the main issues related to the possibility of effectively
exploiting the topical route of administration for copper compounds may be
briefly outlined as follows:
The metal should be complexed by a ligand able to form a stable
and ‘‘shielded’’ lipophilic chelate, thus in vivo allowing its penetration
into the skin, and protecting the ‘‘exogenous’’ Cu(II) ions
from the competition with the numerous potentially complexing
molecules present in the tissue, at least during the diffusion of the
complexes through the skin. This kind of approach could, at
the same time, reduce the risk that ‘‘free’’ copper ions released
from the complex may come into direct contact with the tissues,
causing occurrence of adverse dermatological reactions (170,172).
The ligand also has to be able to coordinate the metal by bonds strong
enough to allow the distribution of the complex in the organism, without
being so stable as to end up with a molecular structure actually
containing copper that is a non-bioavailable. In fact, too stable
chelates, such as those in vivo formed with tetrathiomolybdate
(173,174), are not capable of exerting any pharmacological action
except that of seriously depriving the treated organism of copper.
Both at the local and systemic levels, the ligand has to be nontoxic.
To this end, the natural amino acids and a number of di- or tripeptides,
but also simpler structures such as Cu(II) acetate, appear to
be the favorites, disregarding whether or not they are endowed
with their own anti-inflammatory potential.
Even though the choice of the ligand would have completely fulfilled
the above-mentioned suggestions, it appears possible that many
copper complexes could turn out to be insufficiently lipophilic in
order to permeate intact skin and supply the target tissues with
pharmacologically adequate amounts of the metal. Consequently,
the use of proper vehicles in which to dissolve or suspend the complex
is most likely an important part of the overall task. Aside from
the excipients typically used (i.e., ethanol, dimethyl sulfoxide, etc.),
a very attractive and new approach could be that of embedding the
copper complex in liposomal vesicles (175). As a matter of fact,

The Role of Copper in Inflammation 203
recent papers have shown the high efficiency of such carriers for the
percutaneous administration of a variety of drugs, such as asthma
medications, kanamycin, and indomethacin (176–178).
COPPER ANTI-INFLAMMATORY ACTIVITY: HYPOTHESES
EXPLAINING THE POSSIBLE MECHANISMS OF ACTION
The essentiality of copper for humans was first recognized early in the past
century, when Hart and coworkers (179) showed copper to be critical for
erythropoiesis in the rat. Since then, it became apparent that, during their
evolution, all the aerobic living systems have recruited specialized copper
sites to provide an adequate electron transfer reactivity to the proteins destined
to cope with oxygen (180,181). The facts that in vivo this transition
metal so easily shifts from the Cu(II) to the Cu(I) oxidation state and vice
versa as well as its ability to form stable complexes with electron-donor biomolecules
are the basic reasons for the utilization of copper in the living
oxygen-dependent organisms (182). Thus, copper is a crucial constituent
of many redox enzymes as well as nonenzymatic biologically active molecules,
the importance of which in maintaining physiological homeostasis
of biochemical, cellular, and tissue functions is a well-established evidence.
Table 16 lists some examples of copper-molecules, separated into two
groups, depending on their prevailing enzymatic or nonenzymatic activity.
Inflammation is a physiological reaction, aimed at defending the
organism against exogenous as well as endogenous attack, and copper is
deeply involved in this process. However, inflammation develops through an
extremely complex and strictly interdependent network of single responses,
in which, step by step, different cell types (such as mast cells, polymorphonuclear
leukocytes, monocytes, endothelial cells, macrophages, lymphocytes,
fibroblasts, etc.), and different mediators (such as, histamine, serotonin, arachidonic
acid derivatives, nitric oxide, cytokines, oxygen free radicals,
chemokines, complement factors, etc.) come progressively into play (200).
Copper was shown to be intimately involved in this overall inflammation
network, influencing the behavior of many inflammatory cells, as well as the
metabolism and/or activity of numerous inflammatory mediators (200).
However, providing a comprehensive treatise on this issue would go beyond
the actual purposes of this review; consequently, comments are brief, outlining
some representative examples of the possible mechanisms of action
on which the anti-inflammatory activity of endogenous and/or exogenous
copper may be based.
Histamine Release and Activity
It is generally accepted that histamine is the first chemical mediator to be
released after the inflammatory stimulus, and it is also well established

204 Milanino
Table 16 Examples of Copper-Dependent Molecules Involved in Maintaining
Physiological Homeostasis and Ensuring Adequate Copper Management
Copper molecules In vivo main functions References
Enzymatic proteins
Cytochrome c oxidase Terminal enzyme of the
mitochondrial respiratory
chain
17, 183
Cu,Zn superoxide
Dismutation of superoxide 17, 184
dismutase (SOD 1)
anions
Ceruloplasmin Oxidation of Fe(II) in plasma;
distribution of copper to
extra-hepatic tissues;
antioxidant activity
17, 185–187
Lysyl oxidase Cross-linking of collagen and
elastin in tissue regeneration
153, 188
Monoamine and
Oxidative deamination of
184, 189
diamine oxidases
histamine, serotonin, etc.
Dopamine b-monooxygenase
Catecholamine biosynthesis 190, 191
Monophenol monooxygenase
(tyrosinase)
Melanin biosynthesis 192
Cell surface monoamine Regulation of glucose uptake 193
oxidase
and cell adhesion
Peptylglycine a- amidating Bioactivation of neuroactive 184, 194
enzyme
Nonenzymatic proteins
peptides
Metallothioneins (MTs) Copper storage and detoxification;
stress proteins
195
Transcription factors (Mac 1, Regulation of gene transcription 182
Amt 1, Ace 1, etc.)
for SOD 1, catalase, MTs, etc.
Copper chaperones (Atox1, Copper handling within
196–198
hCCS, hCox17, etc.), and different pro- and eukaryotic
membrane-copper pumps cell types;
(P-type ATPases)
maintenance of copper
homeostasis
A-domains of the clotting Participation in blood
182, 199
factors V and VIII
coagulation process
that its main role in inflammation is to increase the vascular capillary bed
permeability (201–203). According to some early research, a binuclear,
hydroxyl-bridged Cu(II) complex is the active form of histamine (105). On
the other hand, copper, as cofactor of the diamino oxidases, is essential
in the process of histamine degradation (184,189). Furthermore, the metal
seems to regulate the release of this bioactive molecule from the mast cells,

The Role of Copper in Inflammation 205
after their stimulation by a wide variety of proinflammatory noxa (92).
Nevertheless, other biological pathways, e.g., the triggering of the complement
cascade, and bioactive molecules, e.g., the arachidonic acid products and nitric
oxide, are know to play a very significant role in regulating the inflammationinduced
increase of vascular permeability (31,67,203–205). Note that, in all
the above-mentioned processes, some copper-dependent molecules may be
involved directly or indirectly. Therefore, the ambivalent role of copper in
the processes of histamine release, activity, and catabolism, has a yet unclear
relevance in justifying the mechanism of the anti-inflammatory action of this
transition metal, either endogenous or exogenous.
The Arachidonic Acid Cascade
The products of the cyclo-oxygenase pathways, i.e., prostacyclins, prostaglandins,
and tromboxanes, are, since the late 1970s, well-known mediators
of many different biological reactions (31). As far as inflammation is concerned,
the prostaglandins among these molecules are likely to play an
important role, and the inhibition of cyclo-oxygenase (COX-1 and COX-2)
activities actually represents one of the foremost mechanisms of action of
the large majority of NSAIDs (206,207). As previously noted, copper(II),
used as simple salts such as CuSO4 and Cu(NO3)2, seems to be able to interact
in vitro with the activity of these enzymes, altering the equilibrium in
the production of the prostaglandins E2 and F2a, thus promoting an antiinflammatory
effect (28,29). These early observations were later confirmed
using rabbit kidney medulla slices, and rat peritoneal macrophages (208,209).
Nonetheless, as described studying the severe copper-deficient rat, it was
observed that metal deprivation did not have significant effects on either
the lung COX-1 and/or COX-2 activity(ies), or on the reactivity of the copper-deprived
gastrointestinal tissues to the exogenous administration of
prostaglandin F2a and E2 (26). This, in turn, seems to suggest that endogenous
copper might not play a direct biologically significant role in the
arachidonic acid cascade. Moreover, the administration of some copper(II)
complexes with non-anti-inflammatory ligands, such as the bis-pyridine,
bis(2,4-dimethylpyridine), and bis(2,4,6-trimethylpyridine), showed to have
a remarkable anti-inflammatory activity in vivo, but no effect on the prostaglandin
biosynthetic pathways (210). Conversely, Cu(II)2(aspirinate)4 and
Cu(II)2(indomethacinate)4 were reported to decrease the activity of cycloxygenases
to a greater extent compared to the parent ligands (211), thereby
strongly limiting the production of the arachidonic acid different endproducts,
prostaglandins included (212). Thus, in view of the conflicting results
described above, and considering also that the biosynthesis of the various
arachidonic acid cascade products is qualitatively as well as quantitatively
different in the different body tissues (207), whether or not the endogenous
Cu(II) may exert a significant part of its physiological anti-inflammatory

206 Milanino
effects also by regulating the arachidonic acid pathways, is an issue that
still remains to be better clarified.
Connective Tissue Metabolism
Inflammation can be controlled by stimulating the repair processes that
ultimately replace damaged tissue with new cells and extracellular matrix;
when these processes are impaired, the inflammatory reaction may tend to
perpetuate and worsen, eventually degenerating into chronic disease (153).
The roles of the copper-dependent enzyme lysyl oxidase in the biochemical
pathways of reconstruction of collagen and elastin, as well as the participation
of the GHK-Cu in the process of tissue repair and remodelling, were
already mentioned briefly (153,158). In particular, the above copper(II)
tripeptide complex has been the subject of numerous investigations, which
highlight many interesting features of this molecule: (i) GHK-Cu is normally
present in human plasma, probably being in a dynamic equilibrium with its
‘‘de-coppered’’ form GHK; (ii) both GHK and GHK-Cu are generated, in
situ, by the proteolysis of inflammatory and extracelluar matrix proteins
(due to the action of lytic enzymes released by activated phagocytes) during
episodes of tissue damage; (iii) GHK-Cu has been found to be a potent chemoattractant
selective for macrophages, monocytes, and mast cells, but
has no activity in favoring the migration of other inflammatory cell types,
such as neutrophils; (iv) GHK-Cu has a high superoxide dismutase activity,
which contributes to the detoxification of the superoxide anions produced,
particularly in the initial phases of inflammatory response; and (v) in the
inflammation-damaged area both GHK and, especially, GHK-Cu induce
the removal of tissue debris, capillary growth, differentiation as well as viability
and axon outgrowth of neuronal cells, and, finally, the production of
fibroblast-mRNA for the synthesis of matrix protein, such as collagen, elastin,
proteoglycans, glycosaminoglycans, and decorin (158,213) (see also the
website: http://www.skinbiology.com/copperpeptideregeneration.html). Thus,
the evidence reported above seems not only to stress the remarkable biological
importance of the endogenous copper in regulating the tissue repair
process, but may also stimulate research aimed at finding out other copper
peptides suitable to bind copper and able to act in vivo as new antiinflammatory
and, especially, antiarthritic drugs.
Regulation of Nitric Oxide Synthase Activity
In vivo the enzyme nitric oxide synthase is devoted to the production of the
highly bioactive nitric oxide molecule (N¼O). The physiological roles of
nitric oxide are also expressed in decreasing the vascular tone, decreasing
the platelet adhesion and aggregation, and increasing the cytotoxic activity
of macrophages and neutrophils (205,214). Moreover, it has recently been
shown that, in vitro, most of the noxious effects of interleukin-1 (IL-1) on

The Role of Copper in Inflammation 207
the cartilage metabolism (i.e., decrease synthesis of extracellular matrix
component, abnormal cell renewal, enhanced sensitivity of chondrocytes
to oxidative stress, etc.) are probably mediated by the action of superoxide
on nitric oxide that, in turn, acts on IL-1-stimulated chondrocytes (215).
The authors speculated that a combined therapy with NO synthase inhibitors
and antioxidant, e.g., Cu(II)2(3,5-DIPS)4 that seems to posses both
activities, may be promising for full cartilage protection (215). The inflammatory
reaction is one of the conditions in which an upregulation of nitric
oxide synthase has been reported (205,216). It is noteworthy to remark that
the anti-inflammatory agent Cu(II)2(3,5-DIPS)4 has been shown to have a
significant downregulatory activity on the nitric oxide synthase in vitro,
which would be a mechanism well compatible with the pharmacological actions
of this complex (217,218). Moreover, in vitro copper selectively inhibits the catalytic
activity of the constitutive nitric oxide synthase I in C6 glioma cells (219).
Whether or not a regulatory role of endogenous copper on the activity of the
nitric oxide synthases could also occur in vivo, is, at present, unclear. However,
it has recently been observed that the copper-dependent monoamine oxidases
seem to modulate the expression of the macrophage-inducible isoform II of this
enzyme (220), which leaves this intriguing possibility open.
Leukocyte Activity and Migration
The activity of polymorphonuclear (neutrophils) and mononuclear leukocytes
(simply referred to as PMNLs) plays a pivotal role in the development and
control of both acute and chronic inflammations. Among other functions,
the intense phagocytic activity of these cells is specifically oriented towards
eliminating deleterious materials from injured tissue, such as bacteria and other
microorganisms, neoplastic cells, cell debris, antigen–antibody complexes, nonbiotic
foreign particles, etc. (221). Immediately following the noxa the vascular
permeability increases locally, chemotactic/activating factors are released by
the endothelial vascular and inflammatory cells (that are also resident in situ),
as well as by the invading organisms and/or their degradation products, and,
consequently, impressively high amounts of PMNLs are recruited at the
inflamed site from blood (222). Following activation by the different chemoattractants,
the circulating leukocytes are induced to penetrate into the injured
tissue, where they are further activated by a variety of locally present molecules,
and, consequently, prompted to carry out their typical defense functions,
among which the microcosm of biological pathways expressed by the so-called
‘‘respiratory burst’’ plays a primary role. Actually, both endogenous and exogenous
copper are certainly involved in many different steps of the above chain
of events, as well as in the overall inflammatory process (Table 17).
During the PMNLs phagocytosis that occurs in the inflamed area,
these cells dramatically increase (10–15-fold) their oxygen consumption
within a few seconds after contact with the stimulating substance (respiratory

208 Milanino
Table 17 Some Examples of Copper-Containing Molecules Involved in the
Scavenging of Oxygen-Derived Free Radicals in Chemotaxis, Migration and Oxidative
Burst of Polymorphonuclear (Neutrophils) Leukocytes
Compound
Activity on oxygen radicals
scavenging, and other
PMNLs reactions References
SOD 1 Scavenging of superoxide 223
Cu(I)-thionein Scavenging of superoxide and
hydroxyl radicals
127, 224
Ceruloplasmin Scavenging of oxygen radicals 225
Cu(II)-GHK Scavenging of superoxide,
chemotactic activity, and
stimulation of repair processes
158, 213
Cu(II)SO4; Cu(II)-H-
(1-His-Gly)2OH
Scavenging of superoxide 226
Cu(II)-rutin Scavenging of oxygen radicals 227
Cu(II)-bis-pyridine; Cu(II)- Inhibition of superoxide
210
bis(2,4-dimethylpyridine);
Cu(II)-bis(2,4,
6-trimethylpyridine)
production
Cu(II)2(3,5-DIPS)4-albumin Scavenging of superoxide;
inhibition of superoxide
production
139, 140, 228
Cu(II)-salicylate a ;
Scavenging of superoxide 229
Cu(II)-acetylsalicylate a
Cu(II) (salsalate)2 a
Scavenging of superoxide 230
Cu(II) 2(3,5-DIPS) 4;
Cu(II)2(aspirinate)4 a ;
Cu(II)2(indomethacinate)4 a
Inhibition of chemotaxis,
231, 232
migration, and
superoxide production
Cu(II)-piroxicam a
Scavenging of superoxide;
inhibition of migration
128
Cu(II)2(niflumate)4 a
Inhibition of chemotaxis;
superoxide production
233
Cu(II)2(dimethylsulfoxide)2-
(mu-niflumate)4 a
Inhibition of superoxide
140
production
Cu(II)-carbonate in the diet Inhibition of ex vivo adhesion 63
a
Activity of the copper complex is significantly greater than that of the parent ligand used in the
experiment.
Abbreviations: PMNLs, polymorphonuclear (neutrophil) and mononuclear leukocytes, SOD 1,
cytoplasmic Cu, Zn superoxide dismutase Cu(II)-GHK, glycyl-L-histidyl-L-lysine-Cu(II);
DIPS, diisopropylsalicyclic acid.
burst) (234). Inside the PMNLs phagosomes, the enzyme NAPH oxidase
utilizes oxygen to give rise to large amounts of superoxide anions, which,
in turn, leads to the formation of much more reactive oxyl radicals, such

The Role of Copper in Inflammation 209
as singlet oxygen, hydroxyl radical, and hydroperoxyl radical, as well as
hydrogen peroxide (155,234). This sustained production of noxious oxygen
products (that are collectively called ‘‘reactive oxygen species,’’ ROS) has
the obvious scope of opposing the disease-inducing actions of the ingested
particles (either biotic or nonbiotic) during the physiological process of
inflammation (235). However, the oxygen radicals, which the PMNLs’
respiratory burst has generated, could be cytotoxic not only for the foreign
inflammatory material but also for the PMNLs themselves, as well as for
the adjacent tissue cells and matrix or interstitial- and exudate-fluid
biomolecules (235,236). It has been proposed that the chemically nonspecific
character of the ROS-induced reactions affects the cell membranes and the
soluble or matrix biological structures, damaging them and also leading, at
least theoretically, to the extemporary formation of endogenous antigens
that could, in turn, favor the evolution of the acute inflammatory reaction
toward its chronic phase (237). An early study showed that the attack of
superoxide anions on the phagocytic cell membrane might initiate the
arachidonic acid cascade (238). Furthermore, it was observed that the enzymatic
oxidation of arachidonic acid, which ends with the formation of its
active metabolites, generates free radicals, the hydroxyl radical in particular,
as side products (239). In addition, the enzyme nitric oxide synthase may
function to produce superoxide anions (205,240). Thus, a very relevant goal
to be achieved for keeping inflammation under proper control appears to be
the inactivation of the fairly large excess of ROS that may be released within
the inflamed tissue. The enzyme that in vivo is responsible for scavenging
superoxide anions is the SOD 1 molecule. This enzyme, however, not only
is specific for superoxide but is also largely contained only inside the intact
cells; as a consequence, it cannot counteract the other oxygen radicals, and
its action outside the cell is probably a secondary one. Nevertheless, other
natural copper-containing molecules, such as ceruloplasmin, Cu(I)-thionein,
and Cu(II)-GHK, which may be present in the plasma-exudate and/or
in situ produced during inflammation, could efficiently replace SOD 1 in
scavenging superoxide as well as, at least indirectly, the other ROS (Table
17) (155). Interestingly, when inflammation is localized in the skeletal muscle,
the endogenous carnosine and homocarnosine, which may easily bind
copper ions in situ forming stable complexes, are able to act as oxygen radical
scavengers. This hypothesis was proposed on the basis of the evidence
that both Cu(II)-carnosine and Cu(II)-homocarnosine can dismutate the
superoxide anions released from the neutrophils activated by in vitro contact
with phorbol miristate acetate (PMA) (241). It may be relevant to stress that
all the above-summarized evidence could well explain the reason for the
observed copper accumulation within the inflamed tissues of both acutely
and chronically inflamed laboratory animals and humans, previously
described in this review. Also, many small molecular weight copper complexes
exogenously administered have been shown to disproportionate

210 Milanino
superoxide anions and/or inactivate other oxygen radicals (Table 17)
(218,242). Among these molecules are included structures such as some
oligopeptides (not necessarily of endogenous origin), macrocyclic tetraanhydroaminobenzaldehyde,
ethylenediaminetetra-acetate, as well as the
NSAID piroxicam, which have all been shown to be unable to display
any scavenging activity when tested noncomplexed with copper; the complexes
between Cu(II) and all the above listed molecules are, in contrast,
very active superoxide (or ROS) scavengers (128,227,243–245). Conversely,
many other NSAIDs do show an SOD-mimetic activity of their own; this
activity, however, is significantly and, sometimes, dramatically enhanced by
their complexation with copper(II) ions (Table 17). Another biochemical
trait that a relevant number of NSAIDs share with a group of pyridine
derivatives, and which may be related to their mechanism of action, is
the ability to inhibit the PMNLs respiratory burst, reducing the oxygen uptake
of these cells and decreasing their superoxide and other ROS production; once
again, this effect is significantly increased when the same substances are evaluated
as Cu(II) complexes (Table 17) (218). Notably, a study by Nilsson (246)
has shown that the inhibition of the respiratory burst by two copper compounds,
i.e., the Cu(II)2(3,5-DIPS)4 and the Cu(II)SO4, may be primarily due
to the modulation of the protein kinase C activity. According to Sorenson
(107,218), this observation may be extended to other copper complexes of
NSAIDs, such as Cu(II)2(aspirinate)4, Cu(II)2(indomethacinate)4, etc.; moreover,
interference with the protein kinase C activity pathways of action also
accounts for the inhibition of chemotaxis and/or migration shown by the above
(and possibly other) Cu(II) complexes (Table 17).
The crucial process of migration of circulating leukocytes into the
inflamed site, which is restricted to the postcapillary venules of the affected
area, is a complex network of cellular adhesion molecule interactions that
involves also many inflammatory chemoattractants and mediators (either of
‘‘self’’ or ‘‘non-self’’ origin) such as histamine, cytokines (IL-1, IL-6, tumor
necrosis factor a, etc.), chemokines (IL-8, eotaxin, fractalkine, RANTES
proteins, etc.), leukotriene B 4 and possibly other arachidonic acid derivatives,
Cu(II)-GHK, platelet activating factor, complement factor C 5a, nitric
oxide, ROS and oxidized low-density lipoproteins, bacterial lipopolysaccharides,
bacterial formilated peptides, etc. (213,247,248). All these
substances govern, in a time- and stimulation-mediated expression and
activity, the different endothelial and inflammatory cells responses, which
are aimed at creating the proper conditions for the recruitment and activation
at the inflammatory site of the different cells types actually involved in
the physiological onset, development, and control of inflammation. Summarily,
the PMNLs migration can be divided into three distinct phases:
The first one, which immediately follows the insult by the phlogistic
agent, is the tethering of the flowing leukocytes to the

The Role of Copper in Inflammation 211
endothelial vascular cells. This event is caused by the exposure on
the outer surface of PMNL plasma membrane of the adhesion
molecule L-selectin that strongly, but provisionally, binds to a yet
unidentified receptor located on the endothelial cell surface (249).
After tethering, the leukocyte begins to ‘‘roll’’ along the blood
endothelial vessel, to eventually firmly adhere to the endothelium
itself before migrating into the inflamed site. The process of
rolling appears to be basically mediated by the expression of the
endothelial surface molecules P- and E-selectins that bind to
selectin-specific leukocyte-surface ligands, the best known of which
is the P-selectin glycoprotein ligand-1. Arrival of P- and E-selectins
on the scene has the effect of initiating and continuing the leukocyte
rolling, then gradually slowing down, thus preparing the last phase
of the entire migration process, i.e., the firm adhesion to the endothelial
cell of the PMNLs and their subsequent extravasation (249).
The processes of firm endothelial leukocyte adhesion and leukocyte
transmigration towards the inner tissues of the inflamed site are
also extremely complex and not yet fully understood. They imply
the exposition on the leukocyte plasma membrane of a number of
b-integrin adhesion molecules, in particular the b2-integrins LAF-1
(CD11a/CD18) and Mac-1 (CD11b/CD18), as well as, possibly, of
the L-selectin again (250,251). The structures mentioned above
bind to some counterpart endothelial-expressed adhesion molecules
such as the family of intercellular adhesion molecules (ICAMs),
of which the ICAM-1 is the most relevant member, the vascular
adhesion molecule-1 (VCAM-1), as well as the vascular adhesion
protein-1 (VAP-1) (250–252).
It has been proposed that the expression and activation of VAP-1
could be the initial step in the pathway of firm leukocyte adhesion and
transendothelial migration, at least in conditions of a blood flow velocity
comparable with those that allow the binding of other adhesion molecules
(251). In fact, in the absence or blockade of this protein in vitro, the normal
interactions between the leukocyte adhesion molecules, such as the LAF-1,
Mac-1, etc., with their endothelial counter-receptors, such as the ICAM-1,
VCAM-1, etc., are remarkably prevented (50% inhibition), and the leukocyte
transmigration significantly reduced (251,253). Recently, the above
observations have been confirmed in vivo, showing that the deficiency or
the blocking of VAP-1 remarkably inhibits the PMNLs from migrating into
the inflamed sites, thus preventing the customary development of both acute
and chronic inflammations in the mouse (254,255). A very relevant feature
of the VAP-1 is that this endothelial protein is, at the same time, an
adhesion molecule and a copper-dependent enzyme, i.e., a semicarbazidesensitive
amine oxidase, also known under the acronym AOC3 (193,252,253).

212 Milanino
The above biological activities of VAP-1 are exerted by two distinct extracellular
domains of the protein, and are strictly interdependent since the
specific inhibition of the amine oxidase activity also abolishes the ability of
VAP-1 to bind to its, yet unidentified, leukocyte counter-receptor (193,252).
Apart from the copper dependency of VAP-1 physiological role, it already
was reported that the biological action of some inflammatory chemoattractants
and mediators, such as histamine, arachidonic acid derivatives,
GHK-Cu, nitric oxide, and ROS [that also cause the generation of the oxidized
low-density lipoproteins (247)], was modulated by either endogenous
and/or exogenous copper. Moreover, nontoxic amounts of copper sulfate or
chloride have been shown to induce the expression of ICAM-1 on the
surface of chondrocytes in situ in cartilage explant cultures; however, these
copper-induced ICAM-1 molecules failed to promote the adhesion to IL-1activated
peripheral blood monocytes (256).
Another interesting evidence came from studies on the in vivo
lysozyme secretion and ex vivo adhesion and superoxide anion production,
by neutrophils isolated from rats fed a standard or a 200 ppm coppersupplemented
diet, either healthy or affected by complete-adjuvant-induced
arthritis (63). Notably, the lysozyme concentration was measured directly in
the blood of the experimental animals, whereas the neutrophil adhesion and
superoxide generation were evaluated in an ex vivo assay that utilized fetal
bovine serum-coated plates, and exogenously added PMA as stimulant (257).
The results showed that: (i) the dietary supplementation with copper did not
change the blood lysozyme concentration, as well as the adhesion and superoxide
production by the neutrophils isolated from the noninflamed rats;
(ii) conversely, all the above parameters significantly increased in the adjuvant-arthritic
animals of both dietary-treated groups; and (iii) nevertheless,
albeit lysozyme secretion and superoxide generation were found comparable
to those measured in the nonsupplemented arthritic rats, the ability of the
‘‘inflamed’’ copper-supplemented neutrophils to adhere to the assay plates
was markedly reduced (47%; P < 0.001) (Fig. 6). This last observation seems
to suggest that exogenous copper, orally administered by means of a copper
carbonate-integrated diet, could specifically and significantly impair the expression
and/or functioning of the neutrophils’ adhesion molecules, which, in
turn, may partly account for the antiarthritic activity of the 200 ppm coppersupplemented
diet reported in the same experiment (63).
Immune System Development and Reactivity
Research mostly coming from trace element depletion studies clearly
established the importance of an adequate copper (as well as iron, zinc,
and selenium) dietary intake for protection of animals and humans
in the process of host resistance to the invading pathogens (Table 18). In
fact, a large body of concordant data support the following general

The Role of Copper in Inflammation 213
Neutrophil
adhesion
(%)
40
30
20
10
0
*
Diet 30 d Diet 44 d Diet 58 d
AA inoculum AA 14 d AA 28 d
AA not suppl.
AA Cu suppl.
Healthy Cu suppl.
Healthy not suppl.
Figure 6 Effects of normal (Cu ¼ 5.0 ppm) or supplemented (Cu ¼ 200 ppm) copper
diet on the ex vivo adhesion of neutrophils isolated from either healthy and adjuvantarthritic
rats. Percent of ex vivo adhesion of blood neutrophils isolated after 30 days
(AA inoculum), 44 days (AA 14 days), and 58 days (AA 28 days) of feeding, from rats
kept on normal- (Cu ¼ 5.0 ppm) and copper-supplemented (Cu ¼ 200 ppm) diets. Statistics
(Student’s t test): P < 0.01, comparison between Cu supplemented, AA 14 and
28 days, versus not supplemented, AA 14 and 28 days. Note that the adhesion percentages
of neutrophils isolated from both Cu-normal and Cu-supplemented arthritic rats
are significantly different (P < 0.01) from those measured in the respective control
groups (i.e., normal- and Cu-supplemented healthy animals). Source: From Ref. 63.
conclusions (258): (i) an insufficient copper supply is associated with the
impairment of numerous activities of cells involved in both innate and
acquired immune reactions; (ii) the observed alterations may be due to
decreased activities of individual cells, reduction in the total number of
active cells, or to a combination of the above two effects; (iii) the extent
of the damage to the immune system could be sufficient to increase the host
susceptibility to the exogenous (or endogenous) aggressions; and (iv) all
negative effects of copper deficiency on immune reactivity can be reversed
by restoring the normal copper status in the examined subjects.
The evidence reported in Table 18 shows that an analytically measurable
copper deficiency status impairs the overall inflammatory reactions,
decreasing not only the phagocytic-cell ability to efficiently destroy the
invading hosts but also seriously affecting the physiological activity of the
whole battery of immune-competent organs (such as the thymus, bone
*

214 Milanino
Table 18 Some Representative Examples of Dietary Copper-Deficiency on
Mammalian Immunity Functions
Effects of copper deficiency References
Decreased phagocytic and cytotoxic activity of
32
rodent-competent cells
Reduced superoxide anion production and candidacidal
259
activity of animals neutrophils
Increased susceptibility to transplanted leukemia
260
cells in the mouse
Decreased resistance to the attack of different pathogens
261
in the mouse
Impaired ex vivo activity of rat T cells to different stimulants 33
Alterations of protein and lipid composition of murine
262
plasma membrane
Reduced superoxide anion production and candidacidal
263
activity of peritoneal macrophages
Decreased thymus weight and antibody titer in rodents,
264
characterized by a male versus female increased vulnerability
Decreased rat splenic T-cell reactivity to mitogens, and
265
decreased number of helper and cytotoxic T-cell subset
Decreased killing capacity of steer PMNLs 266
Decreased proliferative capacity of rat splenocytes,
267
restored by interleukin-2 or copper addition
Decreased proliferative ability of monocytes and increased
268
number of B-cells in the peripheral blood, in humans
Decreased production of interleukin-2 by human T-lymphocytes 269
Impaired interleukin-2 gene expression in a human
270
T-lymphocyte line (Jurkat cells)
Abbreviation: PMNLs, polymorphonuclear (neutrophil) and mononuclear leukocytes.
marrow, and spleen) and cells (such as the B- and T-lymphocyte cell lines).
Notably, the above effects of copper depletion were significantly more
pronounced in male versus female rats (264). Also, marginal conditions of
copper deficiency (obtained in male rats fed with a 2.7 ppm coppercontaining
diet) that do not involve evident changes of copper levels and
ceruloplasmin activity in the serum nor significantly modify the organ (liver
in particular) copper status are capable of markedly inhibiting the proliferative
response of the splenic mononuclear cells to challenge by mitogens
(271). In addition, the bioactivity of T-lymphocyte-produced IL-2 (which
plays a central role in the regulation of the acquired immune responses)
was dramatically reduced in those experimental conditions (270,271). More
recently, it has been shown that, at least in the Jurkat cells, the decrease
synthesis of IL-2 appears to be caused by the copper deficiency-induced inhibition
of the expression of the NA-AT transcription factor (258,272). Much

The Role of Copper in Inflammation 215
less data are available on the effects that copper supplementation may have
on normal immune reactions. For example, the subcutaneous injection of
Cu(II)2(3,5-DIPS)4 significantly stimulated the production of the different
immune cell lines by the lymphoid tissues and the spleen, thus favoring an
early recovery of injured mice after their exposure to 8 Gy whole-body
irradiation (273). Furthermore, a significant inhibition of delayed-type hypersensitivity
reactions was reported in mice fed with copper-supplemented
diets, and this effect was also shown to be dependent on the amount of
the metallo-element present in the diet as well as on the duration of the
supplemented-feeding treatment (274).
However, the leukocytes, such as neutrophils and monocytes, as well
as the injured endothelia, actively participate in triggering the immune response,
also by means of the production and coordinated action of numerous
inflammatory mediators, such as ROS, nitric oxide, arachidonic acid derivatives,
interleukins, chemokines, adhesion molecules, etc. (213,247,248,256).
As we already stressed, albeit the production of superoxide and other ROS
by tissue phagocytes has an essential role in counteracting the noxious
actions of the invading pathogens, the persistence of an excess of these active
oxygen products may be itself potentially dangerous for the organism. Thus,
it has been previously proposed that an efficient control of the oxygen radicals
generation could be an important goal in ensuring a proper control of
the overall inflammatory reaction development. However, recent data may
appear to challenge the above hypothesis. In fact, it has been reported
that transgenic mice overexpressing SOD 1 showed a significant increase in
delayed-type hypersensitivity reactions; conversely, the selective blockade
of SOD 1 activity by means of disulfiram resulted in a significant decrease
in the development of both delayed-type hypersensitivity reactions and
adjuvant-induced arthritis in the rat (275). In other words, the above results
would suggest that an overexpression of ROS could inhibit the development
of the immune-mediated chronic phase of the inflammatory process, a final
result comparable to that previously described as an effect of severe copper
deficiency in the laboratory animals (26,27,33). Even so, a great number of
copper complexes, both of endogenous as well as exogenous origin, are
endowed with free-radical scavenging potential; furthermore, they are also
able to inhibit the phagocyte respiratory burst, thus lowering the amount
of ROS generated at the inflamed site (Table 17). These copper complexes
have been unequivocally demonstrated to have significant anti-inflammatory
and antiarthritic roles in vivo (Tables 10–13). Moreover, as previously
detailed, the dietary deficiency of copper not only promotes an enhanced
acute inflammatory reaction but also causes a dramatic decrease in the
SOD 1 activity in the total blood cells (22,23). In addition, the development
of acute- and chronic-inflammatory reactions is also critically influenced by
the processes of chemotaxis, endothelial adhesion, and migration of the circulating
inflammatory cells, the immunocompetent ones included (276).

216 Milanino
Interestingly, a number of the copper complexes listed in Table 17 also have
antichemotactic and/or antimigratory effects. Moreover, it has also been
mentioned that the in vitro addition of copper induces the expression of
nonfunctional ICAM-1 molecules on the chondrocyte surface, and the
dietary-administration of high amounts of copper significantly impairs
the ex vivo neutrophils’ ability to adhere to the assay plates, as well as
remarkably inhibits the AA arthritis development in the treated rats (63,256).
On the other hand, it has also been shown that the neutrophils isolated from
copper-deficient mice expressed ‘‘ex vivo’’ a 50% reduced level of the adhesion
molecule CD11b (i.e., Mac-1) on their surface (277).
In conclusion, the experimentally observed activity of many copper
complexes, which significantly ameliorate the conditions of organisms affected
by chronic pathologies characterized by relevant inflammatory and
immunological components, may depend upon a delicate balance in which
the chemical nature of the copper complex, its actual bioavailability, the site
and, especially, the preferred biological process of intervention, as well as the
phase of development of the disease itself are all concurrently involved.
CONCLUSIONS
The evidence summarized seems to reasonably lead to the following general
conclusions:
The nutritional deficiency of copper significantly impairs the organism’s
ability to develop a normal acute inflammatory reaction to
counteract either biotic or nonbiotic attacks. Notably, this effect is
strictly and simultaneously dependent on the amount of copper
contained in the diet, on the length of treatment, as well as on the
gender of the animals studied, males being remarkably more sensitive
than females to dietary copper depletion.
Both acute and, especially, chronic experimentally induced inflammations
cause a net increase of the total copper in some body
compartments, such as blood, liver, and the inflamed area. This
accumulation of copper seems to be directly correlated with the
severity of the pathology considered; furthermore, with the exception
of the kidneys, it does not appear to involve any comprehensive
and biologically significant redistribution of the metallo-element
between the main body compartments. As a consequence, it is conceivable
that the inflammatory process induces an overall enhanced
requirement for copper, which may be accomplished increasing the
metallo-element absorption/retention and/or decreasing its hepatic
excretion, by the affected organism. It may not be secondary to
underscore that a dramatic accumulation of copper has been reported
also in the inflamed synovial fluid collected from rheumatoid

The Role of Copper in Inflammation 217
arthritic patients, as well as of ceruloplasmin in the human inflamed
periodontal tissue; these observations could imply that the need for
more copper to better cope with the inflammatory pathologies may
also characterize inflammations in humans.
In spite of the previously described inflammation-induced increase
in body copper levels, administration of extra amounts of
copper, either by prophylactic feeding of animals with a metalsupplemented
diet or by therapeutically treating them with
exogenous copper salts or complexes, has clearly shown a significant
anti-inflammatory and anti-arthritic potential in vivo.
In general, copper may be considered an anti-inflammatory trace
metal per se, as evidenced by the active participation of the endogenous
metallo-element in the modulation of inflammatory reaction, by the protective
effect of the dietary-supplemented copper on the development of the rat
adjuvant-induced arthritis, as well as by the activity of therapeutically
administered copper, observed regardless of the counter-anion used to carry
it into the body. Nevertheless, the actual effects (i.e., the real potency) of
exogenous copper as an anti-inflammatory and anti-arthritic drug have been
shown to be significantly dependent on the route of administration chosen,
as well as on the ligand used to form the complex. Focusing on the ligands,
molecules that are endowed with their own anti-inflammatory activity,
i.e., a significant majority of the clinically used NSAIDs, appear to potentiate
their effects when administered complexed with Cu(II) ions. However,
albeit a number of data seem to suggest that the observed anti-inflammatory
activity is mainly due to the in vivo action of the intact complexes,
the possibility that some Cu(II)-NSAIDs may also transfer their copper to
one (or more) endogenous ligand(s) cannot be entirely ruled out. As a consequence,
the final results would be, at least partly, obtained by the distinct
effects of the NSAID given, and that of the complex(es) in vivo formed
between the copper ions carried into the body by the drug and some endogenous
ligand(s) of this essential metallo-element. An accurate survey of the
data, which could explain the mechanisms of copper action in modulating
the phlogosis’ development and remission, does not help much in solving
the above problem, neither can it answer the question whether or not the
endogenous and the exogenous metallo-element may somehow overlap in
their anti-inflammatory actions. Actually, the whole scenery is obscured
by the fact that the copper-dependent defense mechanisms (as well as those
of NSAIDs) may simultaneously address different inflammatory pathways;
their targets would also change according to the type of inflammation developed,
as well as to its specific phase of progress. For instance, it has already
been reported that copper has an unclear and, perhaps, secondary role in
regulating the arachidonic acid cascade in vivo, whereas most of the
NSAIDs can strongly inhibit this chain of reactions. Consequently,

218 Milanino
administering a Cu(II)-NSAID complex, the observed influence on prostaglandins,
prostacyclins, and tromboxanes production is, possibly, mainly
due to the effect of the ligand alone. On the other hand, endogenous copper
in the form of SOD 1, ceruloplasmin, Cu(I)-thionein, Cu(II)-GHK, and
perhaps other in vivo existing copper complexes, has remarkable antioxidant
properties. The same has been reported for some NSAIDs, and their
complexation with copper has been described to potentiate these ROSscavenging
effects. It is also well documented that many NSAIDs inhibit
the respiratory burst and, consequently, the oxygen free radicals generation
by activated phagocytes; once again their copper complexes appear to be
more active, but a possible autonomous role for endogenous copper in this
sequence of PMNLs reactions has not yet been recognized with certainty.
The physiological processes of tissue repair and remodelling are known to
be governed by copper-dependent enzymes in vivo (e.g., the lysyl oxidases),
as well as by copper-oligopeptides complexes formed in situ [e.g., Cu(II)-
GHK]; in this case it may be possible that the role of the endogenous copper
is a predominant one, and the administration of exogenous copper might
have the prevailing function of supplying the damaged tissue with the
metallo-element. Finally, it is well established that the pathway of leukocyte
migration towards target tissues represents a key step in the development
and control of acute and chronic inflammation, and it also eventually
involves the activation of the immune response. Some clinically used
NSAIDs have an inhibitory effect on the inflammatory cells’ chemotaxis
and migration, and their Cu(II) complexes clearly show a significantly
enhanced activity. Recently, endogenous copper has been discovered to play
a very remarkable role in the process of leukocyte rolling, adhesion, and
extravasation, being an essential component of the multifunctional vascular
adhesion protein-1, the activity of which appears to be crucial for normal
leukocyte migration. Moreover, addition of simple Cu(II) salts to the assay
medium in vitro promotes the synovial chondrocyte layers to express nonfunctional
ICAM-1 molecules on their surface. Furthermore, neutrophils
isolated from arthritic rats fed with a 200 ppm copper-supplemented diet
have a significantly decreased ability to adhere to the test plates ex vivo,
whereas the ‘‘parent’’ inflamed cells coming from animals fed a diet containing
standard amounts of copper carry out this process normally. Thus,
the examples summarized above clearly suggest that further research is
needed to better understand which one, if any, of the copper-dependent
processes examined could have a predominant relevance in explaining the
natural anti-inflammatory activity of endogenous copper. Moreover, similar
caution is also advisable in speculating on the possible biological pathways
in which the endogenous and the exogenous copper activities may synergistically
overlap.
Finally, a last issue remains to be mentioned briefly, i.e., the potential
toxicity of administered copper, a concern that, unfortunately, is still raised

The Role of Copper in Inflammation 219
by many clinicians not accustomed with the action of copper compounds in
the therapy of rheumatoid arthritis and other degenerative-inflammatory
human diseases. However, existing data appear to clearly indicate that this
concern is not justified. In fact, copper may have noxious effects only following
the chronic oral (or parenteral) exposure to very large amounts of
the metallo-element; in particular: (i) by chronic oral ingestion of foodstuff
(e.g., water) if it supplies the organism with over 5 mg/kg of copper per day;
and (ii) in the case of prolonged hemodialysis with apparatus that cause the
introduction into the circulation of the metallo-element coming from
copper-containing semipermeable membranes or copper tubing (278,279).
Thus, a real risk of copper toxicity may exist only by administering amounts
of the metallo-element certainly far greater than those that may be actually
used for therapeutic purposes. In this context, it seems very relevant to recall
that rheumatoid arthritis patients treated with a full cycle of Permalon TM
did not show the appearance of significant adverse reactions, in particular
none that could be due to iv administered copper (116). This evidence
is noteworthy in that, although the antiarthritic activity of the above drug
cannot be acknowledged in light of modern clinical evaluation protocols,
the absence of significant adverse reactions reported is reliable, being largely
obtained by means of objective measurements.
In conclusion, the research oriented towards the study of the roles
of copper in inflammation, and the utilization of copper compounds as
anti-inflammatory and antiarthritic remedies, is by no means obsolete. In
particular, the chance of targeting the copper preparations directly to the
inflamed sites deserves notable attention. In fact, the percutaneous route of
administration (using liposomes as copper carriers through the skin barrier)
can both minimize the possible, although remote, toxicological hazards, as
well as, especially, favor the accumulation of the metallo-element where its
therapeutic characteristics may be better expressed.
ACKNOWLEDGMENTS
Review of the manuscript by Mrs. E. M. Hostynek-Welch and Dr. J. J.
Hostynek is gratefully acknowledged.
ABBREVIATIONS
ppm part per million (mg/kg)
CPE carrageenan-induced paw edema
CP carrageenan-induced pleurisy
AA adjuvant-induced arthritis
SOD 1 cytoplasmatic Cu,Zn superoxide dismutase
RA human rheumatoid arthritis
sc subcutaneous
(Continued)

220 Milanino
ip intraperitoneal
iv intravenous
Cp ceruloplasmin
NSAID nonsteroidal anti-inflammatory drug
DIPS di-isopropylsalicylic acid
NSN unsubstituted bis(2-benzimidazolyl)thioether
GHK-Cu glycyl-L-histidyl-L-lysine-Cu(II)
PMNLs polymorphonuclear (neutrophil) and mononuclear
leukocytes
ROS reactive oxygen species
PMA phorbol miristate acetate
IL interleukin
ICAM-1 intercellular adhesion molecule-1
VCAM-1 vascular adhesion molecule-1
VAP-1 vascular adhesion protein-1
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260. Lukasewycz OA, Prohaska JR. Immunization against transplantable leukaemia
impaired in copper-deficient mice. J Natl Cancer Inst 1982; 69:489.
261. Jones DG, Suttle NF. The effect of copper deficiency on the resistance of mice
to infection with Pasteurella haemolytica. J Comp Pathol 1983; 93:143.
262. Korte JJ, Prohaska JR. Dietary copper deficiency alters protein and lipid
composition of murine plasma membrane. J Nutr 1987; 117:1076.
263. Babu U, Failla ML. Respiratory burst and candidacidal activity of peritoneal
macrophages are impaired in copper-deficient rats. J Nutr 1990; 120:1692.

The Role of Copper in Inflammation 235
264. Lukasewycz OA, Prohaska JR. The immune response in copper deficiency. Ann
N Y Acad Sci 1990; 587:147.
265. Bala S, Failla ML, Lunney JK. Alterations in splenic lymphoid cell subsets and
activation antigens in copper-deficient rats. J Nutr 1991; 121:745.
266. Xin Z, Waterman DF, Hemken RW, et al. Effects of copper status on neutrophils
function, superoxide dismutase, and copper in steers. J Dairy Sci 1991;
74:3078.
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268. Kelly DS, Daudu PA, Taylor PC, et al. Effects of low-copper diets on human
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and IL-2 mRNA in human T-lymphocytes. J Nutr 1997; 127:257.
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expression is impaired by copper deficiency in Jurkat human T-lymphocytes.
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in vitro activities of lymphocytes and neutrophils from male rats despite minimal
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T-cells mediated by inhibition of the transcription factor NF-AT.
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273. Soderberg LS, Barnett JB, Sorenson JRJ. Copper complexes stimulate hemopoiesis
and lymphopoiesis. Adv Exp Med Biol 1989; 258:209.
274. Pocino M, Baute l, Malave I. Influence of oral administration of excess copper
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276. Jutila MA. Selective lymphocyte migration into secondary lymphoid organs
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277. Percival SS. Copper and immunity. Am J Clin Nutr 1998; 67:S1064.
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Oberleas D, eds. Trace Elements in Human Health and Diseases. Zinc and
Copper. Vol. 1. New York: Academic Press, 1976:415.

10
Copper Jewelry and Arthritis
Brenda J. Harrison
Department of Earth and Ocean Sciences, Copper Research Information Flow
Project, University of British Columbia, Vancouver, British Columbia, Canada
Pseudoscience differs from erroneous science. Science thrives on errors,
cutting them away one by one. False conclusions are drawn all the time,
but they are drawn tentatively. Hypotheses are framed so they are
capable of being disproved. A succession of alternative hypotheses is
confronted by experiment and observation. Science gropes and staggers
toward improved understanding. Proprietary feelings are of course
offended when a scientific hypothesis is disproved, but such disproofs
are recognized as central to the scientific enterprise.
—Carl Sagan (1)
INTRODUCTION
This paper provides an overview, from the point of view of a nonspecialist, of
the ‘‘copper bracelet’’ issue, based on an exploration of the available scientific
literature, popular press, promotional material made available on the
Internet, discussions of the issue of complementary and alternative health
care methods, and books for the lay reader. The focus will be on copper
bracelets, since they are currently the ‘‘in vogue’’ appliances. The discussion
is applicable to other means of wearing metallic copper on the human body,
237

238 Harrison
from copper rings, to copper disks worn under watchbands, or copper
pennies worn inside socks!
‘‘Arthritis’’ is the general term for a suite of over 100 distinct diseases
and conditions. The most common forms are rheumatoid (involving persistent
inflammation of the synovial membranes of joints) and osteoarthritis
(a degenerative process involving the cartilage and bone of joints). Arthritis
affects nearly 43 million Americans (one in six); although arthritis can strike
at any age, aging of the ‘‘baby boom’’ generation will bring an increase in
the number of sufferers in the coming decades (2). Most forms of arthritis
are incurable, but effective interventions (including use of analgesics, steroidal
and nonsteroidal anti-inflammatory drugs—NSAIDs), are available.
They are underused (3).
‘‘The wearing of copper bracelets is a folk remedy for arthritis, but
there are little data to support the efficacy of this remedy’’ (4). The role
of copper in the treatment of arthritis was reviewed by Milanino et al. (5)
to ‘‘stimulate a thoughtful and unbiased reconsideration of the old idea of
using copper as a therapeutic agent in the treatment of those chronic inflammatory
diseases—in particular rheumatoid arthritis.’’
Serum copper is elevated in rheumatoid arthritis although there is evidence
that serum copper does not correlate with disease severity (6–8). From
animal studies it has been established that the rise in serum copper is accompanied
by an increase in liver copper levels (9–12). The modern use of copper
complexes to treat arthritis dates from the 1940s, and the theory that
copper complexes of nonsteroidal anti-inflammatory drugs are more active
and less toxic than the parent compounds is supported by a substantial body
of literature (13,14).
The role of copper in arthritis is complex; the prevailing paradigm
includes the following elements:
1. Copper is an essential trace element; copper deficiency may have
an adverse effect on inflammation and connective tissue disease
such as arthritis.
2. Serum copper is elevated in arthritis.
3. The plasma protein ceruloplasmin, which binds copper, is an
‘‘acute phase reactant’’; the observed elevation of serum copper
is often attributed to an increase in this protein as a result of the
disease.
4. D-penicillamine, used to treat arthritis, binds free copper ions.
5. Copper is an essential cofactor for the enzyme Cu, Zn-superoxide
dismutase; this enzyme neutralizes the destructive free radicals that
may play a role in tissue damage in the disease and it possesses
anti-inflammatory activity.
6. The enzyme lysyl oxidase requires copper as a cofactor and is
required for connective tissue formation.

Copper Jewelry and Arthritis 239
The established role of copper in both superoxide dismutase and ceruloplasmin
offers a framework in which copper could be expected to play a
role in arthritis treatment; however, ‘‘unfortunately, other than for goldsulfhydryl
compounds, there exists a chronic deficiency of solid scientific
information about any role of trace metallic elements, whether they are
administered parenterally, orally, or topically as therapeutic agents in the
management of rheumatoid arthritis (RA) (15). In an intriguing study
using an animal model, a mixture of copper, gold, and silver exhibited significant
antirheumatic function not shown by the individual elements (16).
THE COPPER BRACELET ‘‘MYTH’’ AND HYPOTHESIS
Copper bracelets, armbands, rings, etc., are a folk remedy promoted and sold
for the relief of the symptoms of arthritis. They may be purchased from
pharmacies, health food stores, catalogues, ‘‘new-age stores,’’ and online
suppliers through the Internet. A Google search of the Internet for
(þ ‘‘copper bracelets’’ þarthritis) conducted on January 17, 2005 uncovered
approximately 12,300 Web pages. A Google Scholar search the same day
uncovered only 32 pages using the same search terms. Google Scholar allows
specific searching of scholarly literature (such as peer-reviewed papers,
theses, books, and technical reports). Most of the pages turned up by the
standard Google search are promotional sites, featuring enthusiastic testimonials.
A variety of health-related claims are made on these sites—many claim
therapeutic bracelets date from antiquity and are ‘‘powerful’’ and ‘‘natural.’’
Along with the testimonials, some sites also provide descriptions of the
‘‘theory’’ behind how the bracelets work.
Claims for the Antiquity of the Remedy
Many websites describe the use of copper bracelets for arthritis as having a
very long history, although they are generally vague about how long that
history might be. Many talk of ‘‘ancient writings’’ and ‘‘ancient times’’; or
refer to Roman, Greek, or Egyptian times; or use by ancient Aztecs, Persians,
or Hindus. Others refer to a period of thousands of years, centuries, even
‘‘over 200 years.’’ Some refer to lost knowledge (especially knowledge lost
or abandoned by modern medicine), while others suggest that bracelets have
been in constant use for a very long time.
Claims for the Power of the Remedy
Many websites tell of folklore and ‘‘old wives’ tales’’ that support the idea
that copper bracelets can ease the suffering of arthritis patients. They use
such terms as ‘‘healing power,’’ ‘‘medicinal value,’’ ‘‘ease the pain,’’ ‘‘ease
discomfort,’’ ‘‘relief of pain,’’ or ‘‘increase mobility’’—all of which suggest

240 Harrison
that the bracelets can alleviate or heal arthritis. Many cite the use of copper
bracelets by athletes, especially professional golfers, as evidence of their
effectiveness.
Claims for the ‘‘Naturalness’’ of the Remedy
The ‘‘naturalness’’ of copper bracelets is a prominent feature on promotional
websites. Key phrases include ‘‘natural,’’ ‘‘natural home remedy,’’ ‘‘natural
remedy,’’ ‘‘natural healing,’’ and ‘‘natural relief.’’
Claims About the Underlying Science Behind the Remedy and
Theories About How It Works
Some web pages claim that copper bracelets work by increasing blood or
oxygen flow, or increasing circulation, and thus speeding healing or relieving
pain. Others claim that copper interacts with ‘‘positive fields’’ or energy flow
in the body, or in some way balances electrical potentials. Many claim that
arthritis patients are deficient in copper and that the bracelets supply the
essential element and so promote health and healing; bracelets are promoted
as a worry-free, time-released source of supplemental copper that may
remedy or prevent inflammation. Another group of sites claims that the
copper absorbs toxins by some mechanism or that copper will combat
superoxide radical damage.
Consideration of the Reputed Antiquity of the Remedy
There is a pervasive belief that the use of copper jewelry as a remedy for
arthritis stretches across many cultures and back into history for centuries
or even millennia. The basis for this belief is not clear, although it is likely
that people have worn copper jewelry and amulets since the earliest working
of copper. Arthritis as a condition predates civilization and even the origin of
the human species. Evidence of arthritic deformation can be seen in dinosaur
skeletons and cave bears of the Pleistocene epoch were also afflicted (17).
Neanderthal and Neolithic skeletons, early Egyptian skeletons dating back
to 4000 BC—all show evidence that arthritis was widespread.
The use of copper compounds as therapeutics by early cultures is well
documented and probably dates to the beginning of recorded history and
earlier. Most applications relate to copper’s antibiotic properties (18). The
ancient Egyptian medical text, the Smith Papyrus dating from 1600 to
1300 BC, recounts the use of copper to treat infected wounds and to sterilize
drinking water. Other ancient texts from Egypt, Greece, Rome, the Aztec
empire, ancient India, Persia, and China make it clear that early healers used
a number of copper compounds to treat a wide variety of conditions ranging
from infections to ulcers, inflammation, hemorrhoids, neuralgia, leprosy,
eye ailments, venereal disease, intestinal worms, lung diseases, and in the

Copper Jewelry and Arthritis 241
promotion of wound healing (19). Dresher (20) gives a good, updated overview
of historical and modern uses of copper in medicine.
The Pharmaceutical Journal in 1974 carried a small column called
‘‘Copper on the up and up,’’ which gives an entertaining (and prophetic) history
of the use of copper through the ages and the prospects for its medical
development (21). This brief account includes the statement, ‘‘How long ago
people started to wear copper or bronze ornaments for their supposed magical
or remedial effects is unknown.’’ The discovery that the skeleton of a
Durotrigian defender of Maiden Castle in Dorset who was killed in 43 AD
had been buried with a spiral bronze ring on one of the toes led the author
to speculate that it is ‘‘ ... not too fanciful to suppose that such rings were
employed to ward off the gout or the rheumatism ...’’ This story was
repeated by Dr. John Sorenson (22) in a paper on the therapeutic uses of
copper; however, Sorenson (23), in the next sentence classified copper bracelets
per se as ‘‘modern copper-containing folklore remedies for the treatment
of arthritis.’’ In an e-mail message to the author on November 1, 2004,
Dr. Niall Sharples (24), Senior Lecturer in Archaeology at Cardiff University
and author of an English Heritage book about the history of Maiden
Castle, was able to confirm part of the skeleton story. There is ‘‘ ...indeed
a copper alloy probably bronze ring around the toe of a skeleton from
Maiden Castle. ... These rings are fairly common in the Iron Age of Britain
and they are clearly jewellry worn on both fingers and toes. Burials however,
are less common and there are very few burials with rings if any ... Whether
they also functioned in a medicinal fashion I could not say.’’ The Maiden
Castle book provides a drawing on page 120 of the skeleton; a tankard
had also been buried with the tanker. Spiral bronze rings were common trade
goods of the period and may have been worn as status symbols (24).
Native North Americans have a copper bracelet myth of their own, in
which copper bracelets are worn not for healing, but as a source of mystical
‘‘power’’ (25).
Theophrastus Phillippus Aureolus Bombastus von Hohenheim (1493–
1541), better known as the celebrated Renaissance physician, alchemist, and
philosopher Paracelsus, crafted ‘‘constellation rings’’ in metals that corresponded
to the known planets including copper to bring the protective
influence of Venus and so ‘‘was one of the first recorded copper bracelet
wearers, not to ward off arthritis however, but rather to assure for himself
the benevolence of Venus’’ (19).
As described by Kelly Patricia O’Meara in a Washington Times article
on POWs/MIAs (February 7, 2000), copper bracelets became immensely
popular in 1970 when three American college students began making them
to draw attention to missing-in-action servicemen. The original bracelets
were modeled on a metal bracelet that Robert Dornan (later a Congressman)
obtained from hill tribesmen in Vietnam. Called MIA bracelets, each
had the name of a serviceman missing in Vietnam inscribed on the outside.

242 Harrison
They sold at the time for $3 each, became very popular, and eventually were
worn by 5 million Americans. They are still worn today.
In a 1982 essay, Dr. Gerald Weissmann, NYU School of Medicine,
speculated that the origin of copper adornments as a remedy for arthritis
might have lain with the astrology followers of the 1960s, since the bracelet
wearers Weissmann (26) interviewed talked about ‘‘ ...the healing power of
the metal and the conjunction of the stars ... clear connection between the
planets in their orbit and their metal talisman ... clear faith that gravitational
and electrical forces generated by heavenly bodies were transmitted
to their joints by way of the copper bracelet.’’
It would seem then that the copper bracelet (for arthritis) myth might
have an origin more recent than ‘‘ancient times,’’ and more rooted in superstition
and magic than in any deep, mysterious scientific knowledge. Many
of the ancient texts and new age websites allude to the mystical connection
between elemental copper and the planet Venus.
Consideration of the Reputed Power of the Remedy
For the majority of arthritic conditions (gout is a notable exception), there is
no known cause or cure. There are, however, effective drugs that can slow
the progress of the disease and prevent irreversible joint damage. A number
of ‘‘self-care’’ measures (including relaxation, diet, and exercise) are also
helpful and are routinely recommended by doctors. Copper bracelets are
not among the measures generally regarded as having any effect. Claims
have also been made for a number of dietary supplements, some of which
include copper; however, as of December 1996 the U.S. Food and Drug
Administration (FDA) had not authorized any health claims for any food
or dietary supplement and arthritis (27). This situation remains unchanged
in 2005 according to information on the FDA website.
Consideration of the Naturalness of the Remedy
Copper is a naturally occurring element; a copper bracelet is, of course, a
fabricated object. ‘‘Natural’’ by itself does not confer safety (elemental
arsenic is ‘‘natural,’’ but deadly in a sufficient dose). There is a hypothetical
risk of suffering an accidental injury while wearing a copper bracelet—for
example, an instruction on the Q-Ray Ionized Bracelet 1 website warns
against wearing the bracelets while using an electric blanket ‘‘because the
blanket could heat up the metal bracelet and cause it to burn your skin.’’
Other than suffering from such an accident, the only imaginable risk is developing
a skin reaction to the copper. Hostynek and Maibach (28) recently
conducted an extensive review of available case reports and concluded,
‘‘ ... true allergic reactions to copper appear rare, particularly those induced
by skin contact ...’’ The authors could find very few studies reporting

Copper Jewelry and Arthritis 243
reactions (allergic contact dermatitis and eczema) among copper jewelry
wearers, and it is likely that some of those were actually caused by sensitivity
to nickel, not copper.
Consideration of the Proposed Underlying Mechanism
of the Remedy
In common with materials promoting other unproven remedies (treatments
that have not been shown in repeated scientific studies to work and to be
safe), claims on the bracelet web pages range from the plausible to the
absurd; a survey of the pseudoscience on these pages would make an interesting
paper on its own. Many betray an underlying misunderstanding of basic
science. ‘‘Negative and positive’’ charges and fields echo the pseudoscientific
claims behind magnets and magnet therapy, also in vogue (29). There seems
to be indecision on whether bracelets work by making something flow into or
out of the body. Some sites do cite the scientific literature, although many do
it in a highly selective manner, reprinting a sentence or two to support their
claims. Among the scientific ‘‘facts’’ that are correctly claimed are the essentiality
of copper, its role in superoxide dismutase and antioxidant role,
formation of chelates with substances (amino acids) in sweat, and the possible
role of copper deficiency in the disease. A few sites attempt a balanced
treatment of the current state of scientific evidence and correctly cite supporting
publications but they are the exception.
THE COPPER BRACELET TRIAL
Scientific Evidence for the Use of Bracelets for Arthritis
Therapy Considered
The International Copper Research Association in 1975 published a critical
review of copper in medicine. The authors advocated the use of copper chelates
as anti-inflammatory agents for arthritis treatment. Interestingly, while
copper bracelets were covered briefly, their use was suggested as a means of
exploiting copper’s bactericidal and fungicidal properties:
Finally, it should be kept in mind that people who have a high frequency
of minor skin afflictions of a fungal or a bacterial type,
rather than using copper salts in liniment or salves, may wish to
use the much more convenient metallic form of copper: say in the
form of a copper barrette, a copper chain, or a copper bracelet worn
close to the skin (21). Such a metallic copper device will slowly
release copper ions, most probably in the form of amino acid complexes,
which will permeate both the surrounding skin and eventually
also the body. A maximum figure of the amount of copper released
most likely will not exceed that released from the Copper-T device,
about 45 mg per day. Such a slow, daily release of ionic copper will

244 Harrison
act as a bactericide and fungicide on the skin environment of the
metallic copper. At the same time, it cannot cause any harmful general
effects to the individual, since the amount of copper released
will only constitute a small percent (

Copper Jewelry and Arthritis 245
A word of caution about efficacy—proposed medical therapies must be subject
to three tests: efficacy, or the ‘‘extent to which an intervention does more
good than harm under ideal circumstances’’ (can it work?); effectiveness,
‘‘whether an intervention does more good than harm under usual circumstances
of healthcare practice’’ (does it work in practice?); and efficiency,
‘‘effect of an intervention in relation to the resources it consumes’’ (is it
worth it?) (38). It must be noted that it is not necessary for any therapy
to be fully understood (only that it works).
In 1974, Ray Walker was a university researcher with an interest in
coordination chemistry. For the initial copper bracelets trial, volunteers
with self-declared ‘‘arthritis’’ were recruited by publishing letters in several
Australian newspapers. A substantial number of volunteers (60 of the 300
who answered a questionnaire) were rejected by a physician on the grounds
that they did not fit an arthritic profile (based on an examination of their
questionnaire responses); there was no direct assessment of their health
and so we cannot be certain of the diagnosis of the trial participants (39).
Roughly half reported having worn copper bracelets for arthritis. For the
trial, the remaining 240 volunteers were randomly assigned (randomization
method not specified) to one of three groups: Group I wore a copper bracelet
for one month followed by an anodized aluminum bracelet (placebo) for
one month; Group II did the reverse; and Group III (control) wore no bracelet
for two months. Bracelets were weighed at the beginning and end of the
period and the groups comprised equal numbers of experienced and inexperienced
users. A subjective assessment was made by questionnaire after the
end of the two-month trial—the evaluation categories were ‘‘better’’ (including
both ‘‘much better’’ and ‘‘little better’’), ‘‘same,’’ and ‘‘worse’’ (including
‘‘little worse’’ and ‘‘much worse’’) (35). In the evaluation, a significantly
greater number of the patients who expressed a preference reported that
they ‘‘were better’’ while wearing the copper bracelet than while wearing
the aluminum bracelet. The effect was attributed to the supply of a steady
supplemental amount of copper from the bracelets; copper bracelets were
reported to have lost an average of 13 mg in a month of use and the lost
copper was assumed to have dissolved into the wearers’ sweat and then to
have been absorbed into their skin. Subsequent laboratory experiments
showed that copper from turnings could dissolve into sweat and that copper
complexed to an amino acid perfused cat skin (40,41). The trial results were
reported in preliminary form in 1976, and the data were reported at least
twice again (35–37).
It is difficult to make a confident assessment of what was proved by
the Walker and Keats bracelets trial, and since its publication the study
has been criticized on several grounds:
1. It was poorly controlled (15,42). Because copper bracelets discolor
and turn the wearer’s skin green during use, it is doubtful that the

246 Harrison
experienced users (and even a proportion of the inexperienced
ones) could not distinguish the real thing from the placebo. This
calls into question the ‘‘blinding’’ of the subjects in the trial since
the patient must be unable to distinguish the treatment from the
placebo (43). If either the researcher or patient can guess which
is the real treatment and which is the sham, the study results
may be biased (44). Further, such inadequate concealment in pain
research and complementary and alternative medicine (CAM)
trials results in the treatment effects being overestimated (45,46).
There is no indication in the three papers about the Walker and
Keats trial that the investigators were blinded to the treatment versus
placebo bracelets when weights were taken—in fact there was
no indication that the placebo bracelets were weighed at all (if they
were, no data were presented for them). To establish an effect of a
treatment ‘‘above and beyond natural history of the condition and
non-specific effects,’’ it is essential that the trial is randomized,
controlled and (preferably) double-blinded (44). The Walker and
Keats trial did not meet this standard.
2. The Walker and Keats trial used a vaguely defined outcome (‘‘perceived
effectiveness’’) determined by a subjective evaluation by
questionnaire at the end of each one-month treatment period
and a comparison of the relative effectiveness of the bracelets at
the end of the second period. The data presented are in categories
(‘‘a little worse,’’ ‘‘much better,’’ etc.) but exact criteria for these
evaluations are not specified. It would appear that ‘‘better’’ or
‘‘worse’’ refers to a superficial judgment of the ‘‘effectiveness’’ of
the copper bracelet versus the placebo bracelet, not to the symptoms
of the wearer per se and may be based on the wearers’
appraisal of the ‘‘state of their condition’’ (37). Combining ‘‘a little
better’’ and ‘‘much better’’ into a single category may exaggerate
the apparent effectiveness of the treatment bracelets.
3. In conditions such as arthritis that are characterized by periods of
remission and flare, ‘‘it is not uncommon for two out of every
three patients to report subjective improvement after a treatment
that, unknown to them, contains no active agent’’ (47). There is
a strong suggestion of this in the Walker and Keats (35) data.
A statistical test of the effect of previous experience on the experimental
outcome showed that it was significantly related to the
evaluation provided by the volunteers (35). The largest group of
volunteers overall (30 of 77) perceived no difference in effectiveness
between the copper and aluminum bracelets. The natural
history of their conditions over the two-month trial period could
be expected to significantly affect their perception of the effectiveness
of the bracelet they were wearing during that part of the trial.

Copper Jewelry and Arthritis 247
This phenomenon is common to many pain-causing conditions
and greatly complicates the design of trials in pain research (44).
Moreover, patients often enroll in clinical trials when their symptoms
are at their worst—and ‘‘the next change is likely to be an
improvement’’ (44).
4. The placebo effect, ‘‘a change in a patient’s illness attributable to
the symbolic import of the treatment rather than a specific pharmacologic
or physiological property’’ (44), can be powerful in pain
research. Paracelsus understood the importance of the placebo
effect (or wishful thinking): ‘‘It is not the curse or the blessing that
works, but the idea. The imagination produces the effect’’ (29).
The data for the Walker and Keats trial volunteers who wore no
bracelet at all for the two months (control group) were even more
interesting and suggestive that a strong placebo effect might have
operated in this trial. While 11 of 19 of the previous users said they
were ‘‘worse’’ during the trial period (seven reported ‘‘same’’ and
one ‘‘better’’), the previous nonusers were evenly split, with six
reporting ‘‘worse,’’ six ‘‘better,’’ and 10 reporting no difference (35).
The authors also observed that, of the previous users, 14 dropped
out of the trial because they could not be without their bracelets.
Assuming an initial pool of 40 volunteers in that part of the
control group, seven other testers dropped out for other (unspecified)
reasons. The attrition loss in the ‘‘previous nonusers group’’
was 18—for reasons also not specified in the paper. Did many
‘‘control’’ subjects just get better anyway and so lose interest in
completing the trial? Patient expectations of success of the treatment
are an important component of the placebo effect (44,48)
and highly compliant patients may show better outcomes, just as
they did in the Walker and Keats trial. While all participants were
instructed to carry on normal routines, diet, and medications for
the duration of the trial (35), no data are given on the use of medications
during the trial that might have had a significant influence
on the outcome.
5. Bracelet weight loss was claimed by the authors to supply up to
13 mg/month supplemental copper. It was argued that this copper
dissolved into the sweat beneath the bracelet and perfused the skin
of the wearer. There are some difficulties with the weight loss
claim, however. No error estimates for the calculated mean weight
loss are given in the original Walker and Keats report or subsequent
republications. The bracelets were weighed before the trial
and then sent in plastic bags to the subjects. They were sent back
to the researchers at the end of the trial, ‘‘usually’’ in plastic bags,
at which time they were again weighed. Importantly, an unspecified
number of bracelets were observed to have gained weight over

248 Harrison
the two-month trial period. The researchers took this as evidence
that some bracelets had not been worn—those bracelet weights
and the questionnaire responses of those participants were then
eliminated from the analyses—a substantial source of bias for both
the test and the bracelet weight loss claim. We are not told how
many bracelets were in this group, but of the 160 testers, only
77 wore the bracelets for the full trial, filled out all questionnaires,
supplied classifiable responses, and had bracelets that did not gain
weight. It would have been interesting to know what weight
change was observed in the placebo bracelets and if the trial participants
were able to guess which bracelet (copper or placebo) they
were wearing. In a review discussing the results of the Walker
and Keats trial, the authors admit that the bracelet weight loss
observed might have been caused by mechanical wear, although
in an earlier publication they reported that the used bracelets
did not appear abraded (39,40). Walker et al. (36) also remarked
that the appearance of a green stain on the skin below bracelets
[from copper (II) salt build-up] could be seen as evidence that
some copper must be lost from the bracelet during wear.
6. In a subsequent experiment, copper was shown to dissolve from
turnings that had been submerged and shaken in human sweat
for 24 hours (40). Extrapolating from this result to the dissolution
of copper from a solid bracelet into a thin film of sweat on a
human arm would seem problematic. To begin with, the turnings
present a substantially greater surface area for dissolution compared
with a solid bracelet. Since study participants would have
bathed regularly, it is likely that an undetermined amount of the
copper (green stain) under their bracelets would simply wash off
the skin’s surface and not be absorbed.
7. The trial questionnaires included a semiquantitative assessment of
the diet of study participants. Copper-rich foods (liver, shellfish,
mushrooms, and nuts) were an insignificant part of the diet of
the study participants (35). This is not surprising, since for most
Western diets, these copper-rich sources do not play a large part;
in fact, grains and vegetables commonly provide up to 60% of copper
intake (49). The observation that most of the participants’
diets were in the ‘‘low’’ category for intake of copper-rich foods
is typical of populations as a whole and does not indicate anything
useful about what the overall dietary copper intake of study subjects
might have been—it certainly cannot be taken as robust
evidence that the participants were copper deficient.
Walker and Keats were initially tentative in their support for the therapeutic
value of the bracelets, and suggested in their original paper that the

Copper Jewelry and Arthritis 249
subjects who had not been bracelet wearers before the trial may have been
‘‘less ready to commit’’ themselves. In a 1977 letter in The Medical Journal
of Australia, Whitehouse and Walker (50) mention that the ‘‘Pharmacy Board
in Victoria is currently reconsidering its ban on the sale of copper bracelets’’
and that there had been discussion of the issue in the lay press. It is possible
that people who volunteered for the trial might have felt quite passionate
about the issue before the trial and that their enthusiasm could have affected
the trial’s outcome, as the investigators themselves indicated. In calling for
trials to fully validate the role of the bracelet, the researchers concluded, ‘‘even
if it has little value, except as a placebo, it does have the merit of being of little
harm (by virtue of being the most inefficient source of a potential toxin)’’ (50).
Further well-designed and properly conducted clinical trials, rigorously controlled
for placebo effects, and double blinded, would be necessary to confirm
or refute the findings of the Walker and Keats trial and prove or disprove
finally the claims of the promoters of copper bracelets.
The study of ‘‘the copper bracelet myth’’ and the science behind it led
to the development of a topical anti-inflammatory copper-salicylate gel
(36,37,51) that is available as an over-the-counter product in several countries.
An independent, double-blind, placebo-controlled, randomized trial
of the gel failed to confirm its effectiveness: applied to the forearm it was
no better than the placebo gel for pain relief in patients with osteoarthritis
of the hip or knee (52). The treatment gel also produced significantly more
skin rashes than the placebo. In 1998, The Medical Journal of Australia
published a letter critical of the trial by one of the gel’s developers (and
a colleague of Dr. Walker’s). Dr. Boettcher criticized the gel study on
the grounds that the gel should have been applied (as directed) at or near the
hip or knee and the composition of the gel was incorrectly described in
the article (53). Dr. Boettcher also took exception to the statistical analysis
used. Drs. Brooks and Kellett (54) countered these objections—the trial was
designed to investigate systemic effects on distant joints—and they refuted
Dr. Boettcher’s statistical argument.
Investigation of the effectiveness of topical therapies for arthritis pain
relief continues—results of several substantial studies have been published
recently. A meta-analysis of randomized controlled trials of topical NSAIDs
(popular over-the-counter drugs) used for the relief of osteoarthritis pain
concluded that they are no more effective than a placebo beyond two weeks
of treatment (55). A systematic review of double-blind, placebo-controlled
trials for efficacy concluded, ‘‘topical NSAIDs were effective and safe in
treating acute painful conditions for one week’’ (56).
A New Bracelet Study
A new study of the effects of copper/zinc bracelets on joint and muscle pain
was conducted at the Mayo Clinic and published by them in 2002 (57). The

250 Harrison
study was designed as a randomized, double-blind, placebo-controlled trial
of ‘‘ionized’’ wrist bracelets that were advertised and marketed as effective
for the treatment of pain. Because the bracelets (both ‘‘ionized’’ and placebo)
used in the trial were made mostly of copper (85% copper/15% zinc)
they are relevant to the discussion of the Walker and Keats bracelets trial.
There are interesting similarities between the two studies:
1. The subjects for both studies were volunteers with self-reported
pain (in the Mayo Clinic study they were recruited by posters in
the clinic).
2. The patients in both studies wore bracelets for a one-month test
period.
3. In both studies, patients replied to questionnaires on which (among
other questions) they rated their own conditions subjectively.
4. The placebo effect was evident in both trials.
5. In both trials, a proportion of the patients believed in the power of
the remedy beforehand.
In the Mayo Clinic study, participants were asked in advance if they
believed the bracelets would be effective and, of the participants who
answered the question, 80% replied in the affirmative; however, only 4.4%
reported having ever used a bracelet before the trial. Patients wearing both
placebo and ‘‘ionized’’ bracelets reported statistically significantly lower
pain scores during the course of the trial, but the two groups showed the
same degree of improvement, leading the authors to conclude that the treatment
bracelets were no better than the placebos.
The new study differed in methodology in significant ways from the
Walker and Keats trial. Firstly, the Mayo Clinic study used a more clearly
defined outcome (pain rated on a scale of 1–10) and they evaluated pain at
several points during the month (days 1, 3, 7, 14, 21, 28). Remarkably, on
day 1, 63.3% of the ‘‘ionized’’ bracelet wearers and 68.5% of the placebo bracelet
wearers reported an improvement in maximum pain scores—from the
study’s description, there is good reason to expect that an especially strong
placebo effect may be evidenced. As part of the Mayo Clinic study design,
patients were instructed in the correct placement of the bracelets according
‘‘to the manufacturer’s recommendations’’ (57)—a procedure that very
likely induced additional confidence in the patient that the remedy was likely
to be powerful, judging by the manufacturer’s instructions available on their
website—in particular, the specific instructions for changing the orientation
of the terminals if the bracelet is worn on the left instead of (recommended)
right wrist; the implication of the term ‘‘terminal’’ for the bobble on the
open end of the bracelet; the instruction that the ‘‘terminals’’ should be 1 ⁄2
to 1 1 ⁄2 inches apart during use; and the warning, ‘‘Do not let the terminals
directly touch each other.’’ As Bratton et al. (57) point out, going through
these instructions with patients is an excellent example of the ‘‘healing

Copper Jewelry and Arthritis 251
ritual’’ described by Kaptchuk (48) as producing an ‘‘exaggerated placebo
effect’’ and it could well explain the observation that 2/3 of the participants
reported an improvement after just one day of wearing the bracelets. Interestingly,
although 80% of the participants answering the question reported
ahead of time that they believed the remedy would work, the highest proportion
reporting an improvement in pain scores during the trial was 77.7%.
Unfortunately, the Mayo Clinic study did not have a control group that
wore no bracelet at all during the trial—this could have provided valuable
information about the significance of the pain improvements (which were
not large) reported by both groups of study participants.
In 2003, the U.S. Federal Trade Commission (58), citing the Mayo
Clinic study, charged the marketers of the Q-Ray Ionized Bracelets with
making false and unsubstantiated claims, specifically ‘‘deceptively claiming
that the Q-Ray Bracelet is a fast-acting effective treatment for various types
of pain and that tests prove that the Q-Ray Bracelet relieves pain.’’
HealthScout News reporter Ed Edelson concluded an online article about
the study (HealthScout News, November 12, 2002) with this wry comment
from Dr. Robert Bratton, the study’s lead author: It’s all a placebo effect and
‘‘based on the study results, you may be just as well off wearing a rubber
band around your wrist and saving the money spent on the bracelet.’’
The Gold Ring Study
Gold treatments (by injection) are used in the treatment of rheumatoid
arthritis and an intriguing study published in 1997 provided some evidence
that gold from rings may delay articular erosion in rheumatoid patients (59).
In the study, confirmed rheumatoid arthritis patients were divided into two
groups—non-ring wearers and ring wearers—and their hands and wrists
examined by standard radiographic technique. While the two groups had
statistically indistinguishable overall articular erosion, the fingers of the
ring-bearing hands were significantly less deformed. While the study was
small (55 subjects in total), and, as the authors admit, it is possible that the
effect could relate to the different pattern of use between the left (ringbearing)
and right hand, it does suggest that gold from the rings might
provide a protective effect.
THE PRESENT STATE OF THE COPPER BRACELETS ‘‘ISSUE’’
More Recent Studies Have Been Less Promising or Just How
Satisfied Are Bracelet Users?
Two features of the most common arthritic conditions make arthritis
patients vulnerable to the lure of alternative remedies: their conditions have no
known cause or proven cure and they are characterized by chronic pain (33).
In 1997, American consumers spent approximately $27 billion on alternative

252 Harrison
therapies (60). Alternative therapies are ‘‘aggressively promoted’’ and, in
the opinion of D.M. Marcus (61), MD, Baylor College of Medicine, ‘‘most
AM [alternative medicine] information that is readily available to lay persons
is misleading or wrong.’’ This point of view is supported by a survey
made in May 1998 of Internet information for patients with rheumatoid
arthritis. After evaluating the quality and source of materials offered its
authors concluded, ‘‘over two-thirds of the sites with overt financial aims
promoted the use of alternative therapy, often claiming that their products
were effective for arthritis and other conditions ... Although some alternative
therapies may be efficacious, most have not been adequately studied,
and often, positive studies have major methodological flaws’’ (62).
Surveys of patterns of usage of copper bracelets have been conducted
in Australia/New Zealand, North America, and the United Kingdom. Let
us suppose that it is true that copper bracelets provide great symptomatic
relief for arthritis sufferers. Since they are widely available, inexpensive,
and heavily promoted, one would expect to observe high rates of bracelet
use among patients, and patients should show high rates of satisfaction
(and continued use). There is evidence to suggest that patients are generally
aware of the copper bracelet claims. For example, 75% of the 51 Hawaiian
patients with inflammatory arthropathies in a Hawaiian survey had heard of
copper bracelets as an alternative therapy (63). Findings from this and several
other patient surveys are summarized in Table 1. Patients reported that
they turn to CAMs to relieve pain; many discontinued conventional medications
because they found medication schedules complicated (71).
Patients also perceive the inability of conventional therapies to cure or
adequately control their disease and the potentially serious adverse effects they
may cause as reasons to turn to alternative treatments (72). Well-publicized
Table 1 Use of Copper Bracelets/Bands by Arthritis Patients
Location
No. of
patients using
% of survey
population
Age of patients
(yrs) Citation
Australia 45 61 15–65þ (64)
Australia/
New Zealand
21 70 12.1–27.2 (65)
Australia 3 3 Mean ¼ 61.1 (66)
Canada 4 57 9.3–19.3 (65)
Canada 16 11 Mean ¼ 10.4–10.9 (67)
England 75 38 N/A (68)
England 45 28 N/A (69)
United States 17 13 Adults (70)
United States 42 29 Mean ¼ 55.5 (60)
United States 11 21.6 Mean ¼ 51 (63)

Copper Jewelry and Arthritis 253
side effects of conventional medicine (in contrast with the natural/safe/
gentle claims for CAMs) provoke an ‘‘exaggerated fear of conventional
medications’’ among arthritis patients (61). Sometimes, new studies show that
patients’ concerns about the potential side effects of conventional drug therapies
are justified. For example, in September 2004, it was revealed that the
popular Merck arthritis and acute pain medication VIOXX 1 (rofecoxib), with
worldwide sales in 2003 of $2.5 billion, increased the relative risk of heart
attack and stroke when used for more than 18 months. The press coverage
of this announcement and subsequent negative studies on VIOXX was extensive
and featured such frightening headlines as this one that appeared in The
Times (London) online edition on January 25, 2005: ‘‘VIOXX may have killed
thousandsofUKpatientsstudywarns.’’
CAM users tend to be female, ‘‘better educated,’’ and employed.
Many arthritis patients use CAMs to supplement their conventional treatment
and report that friends and relatives and the mass media are their main
source of information on alternative treatments. In a recent survey of arthritis
patients in New Mexico, for example, the most frequently cited sources of
information on CAMs were family and friends (66.1%), medical doctors
(56.1%), magazines and books (34.6%), radio/TV/newspapers (22.6%), and
the Internet (11.3%) (73).
As for effectiveness, in one survey, 74% of adult Americans polled
expressed a belief that copper bracelets would help treat arthritis; tellingly,
only 13% of arthritis patients reported having used them (70). Several small
studies have reported on the success rate claimed by arthritis patients for
various alternative and complementary therapies (Table 2). Overall, copper
bracelets did not fare well in these surveys. Among the therapies with a
Table 2 Patient Satisfaction Level with Bracelet Use: Proportion of Patients Using
or Having Used Copper Bracelets Reported to Have Found Them to Be ‘‘Helpful’’
Location
Patients reporting a benefit from using bracelets
No. (%)
using
bracelets
No. (% of users)
reporting benefit or
currently using
% total
patient
population n Citation
Australia 45 (61) 6 (13) 8 74 (64)
England 75 (38) 6 (8) 3 199 (68)
United States 11 (21.6) 4 (36) 7.8 51 (63)
United States 42 (29) a
9 (21) b
6 146 (60)
United States N/A (3.9) N/A 612 (73)
a
‘‘Ever used’’ includes both presently using and used in the past.
b
Compare with clinical improvement observed in 30% to 35% of patients treated with a placebo
in clinical drug trials (42).

254 Harrison
higher reported ‘‘usefulness’’ or success rate are chiropractic, herbal therapies,
nutritional supplements, medication/relaxation, electrical stimulators, special
diets (especially avoidance of certain foods), acupuncture/acupressure,
osteopathy, and faith healing. Most unproven remedies, including copper
bracelets, are considered ‘‘harmless,’’ with the caution that even innocuous
therapies could be harmful if diagnosis is delayed (especially with the advent
of aggressive therapy early in the course of the disease that has been shown to
prevent permanent joint damage) or proven therapy neglected (74).
Rao and coworkers (75) conducted a longitudinal study on the same
group of rheumatology patients they reported on in 1999, with follow-up
surveys at six and 12 months. Over the one-year period, three patients
(out of 174) started using a copper bracelet, three maintained their use,
and five stopped using a bracelet. Of the 28 patients in their study who
reported having ‘‘ever used’’ a copper bracelet but had stopped, 24 gave
‘‘did not help’’ as a reason, one claimed to have been made worse by wearing
a copper bracelet (two said ‘‘other’’ as a reason, and one declined to say) (75).
In a recent study, a group of 40 Asian and 40 Caucasian rheumatoid arthritis
patients in the United Kingdom were asked to provide a subjective
ranking of effectiveness of complementary therapies on a five-point scale;
‘‘The two groups rated copper bracelets and magnets very similarly, only
just above useless’’ (76).
Additional Research and Scientific Data Needed to
Resolve the Copper Bracelets Issue
The precise ‘‘ ... role of copper in inflammatory processes remains quite
obscure. Both pro- and anti-inflammatory effects of copper have been documented’’
(15).
The studies by Ray Walker and colleagues were visionary, but inadequate
to establish or refute the efficacy of copper bracelets for arthritis
therapy. The comment is made repeatedly, in the scientific and other literature,
that the available data are not sufficient to constitute a proof of the
effectiveness of externally worn copper as therapy for arthritis. The original
trials could certainly be repeated and improved on by:
recruiting known patients with clearly defined and medically
verified disease;
better screening patients for prior bias about the therapy;
improving placebo design and control;
using objective measures of pain and disease activity as measurable
study outcomes;
rigorously measuring bracelet weight changes during wear, establishing
a rate and accounting for loss of weight due to abrasion and/
or increase in weight, and using a statistically reliable method of
determining weight change;

Copper Jewelry and Arthritis 255
modeling corrosion of copper from bracelets and complex formation
in sweat during use; and
using a properly double-blinded study design.
Recent surveys of the use of complementary and alternative therapies
by arthritis patients do not lend strong support to the belief that bracelets
are found to be of much use and this must call into question any impulse
to do more research on them. As discussed above, convincing evidence that
bracelets exert more than a placebo effect is lacking. A survey of 2146 primary
care physicians and rheumatologists ranked copper jewellery (at 25%)
fifth on the list of ‘‘top 10 alternatives doctors advise against.’’ In a parallel
survey of 790 people with arthritis who read Arthritis Today, ‘‘metal jewellery’’
was an alternative reported as used by only 15% of the survey respondents
(77). A search of the new National Institutes of Health (NIH) Clinical
Trials.gov on October 20, 2004, found 136 ongoing studies on ‘‘arthritis’’
but none involving copper bracelets. The nonprofit agencies and foundations
dedicated to the well-being of arthritis sufferers and governmental regulatory
agencies are similarly unenthusiastic about copper bracelets as a folk
remedy; their position statements are compiled in Appendix A.
On the other hand, there is a definite need to establish the extent of
dermal absorption of copper from a metallic source. ‘‘Dermal exposure to
copper can result from the use of consumer products containing copper pigments,
through the use of copper as an agicide in swimming pools and the use
of copper jewellery. No quantitative exposure levels could be found’’ (78).
There remains a need for objective, stringent human studies involving
copper and arthritis. Several lines of investigation that have been proposed
over the years seemed interesting and yet, to my knowledge, remain to be
fully studied:
1. The role of dietary copper deficiency in the etiology of arthritis and
the use of supplemental copper (or copper-rich diets) in arthritis
treatment:
A small double-blind, double placebo controlled factorial trial
of copper (in a complexed form at 3 mg/day) and/or omega-3
fish oil supplements showed no significant effect of copper on
systemic lupus erythematosus disease activity—a significant
decline in disease activity was demonstrated for the fish oil
supplement (79).
2. Quantification of the supplemental supply of copper from an
intrauterine device (IUD) and an epidemiological study of arthritis
among copper IUD users.
3. The mechanism of formation of copper complexes with salicylates
from arthritis analgesic products in vivo and how that increases
their effectiveness.

256 Harrison
IS THERE LIKELY TO BE A FUTURE FOR COPPER BRACELETS IN
ARTHRITIS CARE?
In Geneva in January 2000, the World Health Organization launched
The Bone and Joint Decade: 2000–2010 (80). In recognition of the immense
burden of the disease, the U.S. Congress initiated a national arthritis prevention
program with a budget of $12 million for the year 2000 (2). One
of its goals was to strengthen the science base for arthritis treatment options,
especially the roles of good nutrition, appropriate exercise, evaluation of
intervention strategies including self-help patient education programs, and
promoting good communication. There is good reason to suppose that the
therapeutic use of copper for arthritis will be a recurring issue. Copper bracelets
are not the only copper product to be touted as therapeutic. America’s
first great medical quack claimed to cure disease with copper rods passed over
the body (81). The Internet is a rich source of unproven and quack remedies
involving copper, and it is likely that more will spring up from time to time
(Appendix B). The Internet makes it much simpler and less expensive for
unproven claims to be widely promoted.
In 1999, the establishment of the National Center for Complementary
and Alternative Medicine (NCCAM) under the U.S. National Institutes of
Health signaled a shift in the American medical establishment’s approach to
alternative therapies. Scientists had been understandably reluctant to risk
ridicule by attempting serious study of therapies not recognized by traditional
medicine. Ray Walker experienced this first hand while embarking
on the investigation of the copper bracelet by publishing the initial invitation
to study participants in the popular press: ‘‘The letter caused some
concern to several of my colleagues and raised the eyebrows of my medical
friends. Although I had some qualms about my action, these were somewhat
allayed by the work of Hagenfeldt ... and Okereke et al.’’ [(37); citing
Hagenfeldt’s 1972 study on the mode of action of the copper-T IUD and
Okereke, Sternlieb, Morell, and Scheinberg’s 1972 paper in Science on the
systemic absorption of copper from an IUD]. NCCAM has become a formidable
force in health research and for 2006 requested a budget of
$122.1 million (82). Its activities include funding research projects and clinical
trials (it identifies conditions affecting the elderly as a priority area for
investigation), developing a comprehensive communications program, and
training young scientists in CAM. In its first five years it supported more
than 1200 projects on various CAMs, including clinical trials on acupuncture,
mind–body medicine, and herbal products.
The NCCAM is not without its critics (e.g., Ref. 83), and the conflict
between conventional and alternative/complementary medicine is a hot
topic in journals such as Journal of the American Medical Association,
British Medical Journal, and the Medical Journal of Australia. The substantial
research award amounts now available for studies on CAMs mean that

Copper Jewelry and Arthritis 257
they are likely to continue to enjoy a public profile, particularly as more
studies are completed, published, and covered in the popular press—as
the example of the Mayo Clinic Q-Ray bracelet study demonstrated. The
methods used to study CAMs are also likely to excite ongoing controversy.
There is even an interesting discussion of whether CAMs should be evaluated
in a fundamentally different way from conventional therapies. The
placebo effect, for example, that played such a prominent role in the bracelet
trials seems to play an important role in many alternative therapies. In a
highly entertaining essay in the Annals of Internal Medicine, Doctor of
Oriental Medicine (OMD) Ted Kaptchuk examines the possibility that there
is some real value for the placebo effect in clinical practice. Kaptchuk’s (48)
essay illustrates vividly a growing gulf between CAM proponents and practitioners
of traditional science and medicine: ‘‘Some may dismiss these types of
investigation as useless. After all, a placebo is just a placebo. Others would
argue that such avoidance impoverishes and narrows the understanding of
what patients receive from alternative medicine ...’’ Examination of complementary
and alternative therapeutic approaches (especially emphasizing
longitudinal studies) is a focus of the new ‘‘National Arthritis Action
Plan’’ (74). There clearly needs to be much better patient education and public
education about arthritis, and there is a definite need on the Internet for
additional high-profile, reputable sites that meet the highest standards of
scientific evidence. This might help to counteract the clamor of the lesscredible
websites.
Considering the scant evidence of the metal’s systemic absorption and
anti-inflammatory effects in laboratory rats, one can safely say that
nothing has been learned in the past 3,500 years that would explain
the longevity of the belief in copper’s powers.
—Sharon L. Kolasinski (84)
APPENDIX A: POSITION STATEMENTS OF SUPPORT
ORGANIZATIONS, GOVERNMENT AGENCIES, ETC.
Arthritis Foundation (USA)
From Arthritis Today magazine (available on the society’s website):
‘‘there is no scientific research proving they provide any
therapeutic benefit for arthritis. Nor is there any research proving
they don’t.’’
From their new guide to alternative therapies: ‘‘Expert Opinion:
Wearing a copper bracelet won’t hurt, but there’s not enough scientific
evidence that it helps’’ (85).

258 Harrison
The Arthritis Society (Canada)
‘‘On the hardware side of the store, things get even stranger:
‘‘inductoscopes’’ (allegedly of magnetic induction), ‘‘solarama boards’’
(to align mixed-up electrons), ‘‘earthboards’’ or ‘‘vitalators’’ (to
produce rejuvenating electrons), ‘‘oxydonors’’ (to reverse the death
process into the life process as a cure for arthritis) and, oh yes,
copper bracelets. None of them has ever been proven effective in
treating arthritis and relieving its pain’’ (86).
Arthritis Foundation of Victoria (Australia)
Under unproven remedies: ‘‘The use of copper has little scientific
support. Copper bracelets are a popular alternative treatment for
arthritis, but there have been no proper trials of copper bracelets
in rheumatoid arthritis’’ (87).
National Institute of Arthritis and Musculoskeletal
and Skin Diseases, NIH (USA)
‘‘Some of these alternative therapies have included wearing copper
bracelets, drinking herbal teas, and taking mud baths. While these
practices are not harmful, some can be expensive. They also cause
delays in seeking medical treatment. To date, no scientific research
shows these approaches to be helpful in treating osteoarthritis’’ (88).
National Arthritis Action Plan (USA)
‘‘Using unproven remedies for arthritis can waste time and money
... may cause people to delay seeking early diagnosis and appropriate
management. Because of their ongoing pain and lack of awareness
of helpful intervention strategies, people with arthritis are particularly
vulnerable to promoters of unproven remedies’’ (74).
Federal Trade Commission—FTC (USA)
‘‘Consumers spend an estimated $2 billion a year on unproven arthritis
remedies—thousands of dietary and so-called natural cures, like ...
magnets and copper bracelets. But these remedies are not backed by
adequate science to show that they offer long-term relief’’ (89).
Better Business Bureau of Eastern Massachusetts,
Maine, and Vermont (USA)
‘‘Quack arthritis cure ... Doctors say uranium is useless against
arthritis, and a copper bracelet does nothing but turn your wrist
green’’ (90).

Copper Jewelry and Arthritis 259
Food and Drug Administration—FDA (USA)
Under unproven remedies: ‘‘Some remedies, such as vinegar and
honey or copper bracelets, seem harmless. But they can become
harmful if they cause people to abandon conventional therapy’’ (27).
APPENDIX B: MISCELLANY
Another Copper Bracelet Myth
Native North Americans recount a charming myth about a girl and a bear.
It exists in several different versions and is a long and richly detailed tale.
Briefly, a girl out gathering food slips on bear excrement (violating the
bear’s mythical power, which is believed to be associated with its excrement).
She utters an oath, further insulting the bear. The bear was considered the
most powerful of animals—and thought to have the mystical powers of a
shaman. The bear kidnaps the girl and leads her to a mystical world where
it keeps the girl as its wife. The version collected at Bella Bella, British
Columbia, includes this lovely detail: ‘‘He asks her what sort of excrement
she has that would give her the right to scold him, and she says her excrements
are abalone shells and copper. He tells her to sit down and show
him, which she does, slipping off one of her copper bracelets’’ (25).
Other Copper-Crafted ‘‘Healing Products’’ Found
On the Internet:
Copper cup (add warm water, let stand for 8–10 hours and drink
the healing liquid).
A healing wand of copper, brass, silver, quartz, and polished gemstones,
with a hollow handle for addition of herbs, etc.
A small copper disk to wear on the bottom of a wristwatch as an
alternative to a bracelet.
Copper patches as an alternative to copper bracelets.
Mineral water enriched with copper (100 ppm) for joint problems.
Copper coil placed in a warm water foot bath to draw toxins from
the body through the feet and so treat a variety of ailments and
restore energy.
In the daily newspaper:
Copper wires to insert in the nose to cure the common cold,
described in a Reuters story carried by The Vancouver Sun on
February 16, 2000.
In the scientific literature:
A copper bed sheet for treatment of the pain of fibromyalgia (91).

260 Harrison
Early Copper Quackery from a Classic Book on Pseudoscience
America’s first great quack was Dr. Elisha Perkins (1740–1799). The
doctor had a theory that metals draw diseases out of the body, and in
1796 patented a device consisting of two rods, each three inches long.
One rod was supposed to be an alloy of copper, zinc, and gold; the
other—iron, silver, and platinum. By drawing ‘‘Perkins’ Patented
Metallic Tractor’’ downward over the ailing part, the disease was
yanked out. Perkins sold his tractors for five guineas each to such
notables as George Washington, whose entire family used it, and
Chief Justice Oliver Ellsworth. His son, Benjamin D. Perkins (Yale,
class of ’94) made a fortune selling the tractors in England. In
Copenhagen, twelve doctors published a learned volume defending
‘‘Perkinism.’’ Benjamin himself wrote a book about it in 1796, containing
hundreds of stirring testimonials by well-educated people.
They included doctors, ministers, university professors, and members
of Congress. Most historians of the subject think the old man actually
believed in his tractors, but that his son—who retired in New
York City as a wealthy man—was simply a crook promoter. It is
worth noting that orthodox medical opinion, by and large, ignored
Perkinism, regarding it as not worthy of serious refutation. One doctor,
however, did trouble to make some tests with phony tractors.
They looked like the genuine article, but actually were non-metallic.
His results, of course, were excellent. Oliver Wendell Holmes, in an
amusing discussion of Perkinism, relates that one woman was quickly
cured of pains in her arm and shoulder by using a fake tractor made
of wood. ‘‘Bless me!’’ the woman exclaimed. ‘‘Why, who could have
thought it, that them little things could pull the pain from one!’’ (81).
Web Resources: Sites with Reliable Information on Arthritis
American College of Rheumatology: http://www.rheumatology.org
Arthritis Care (United Kingdom): http://www.arthritiscare.org.uk/
Arthritis Foundation (United States): http://www.arthritis.org/
Arthritis Foundation of Victoria (Australia): http://www.arthritisvic.
org.au/
Mayo Clinic: http://www.mayoclinic.com/
National Center for Complementary and Alternative Medicine
(NCCAM)(NIH): http://nccam.nih.gov/
National Institute of Arthritis and Musculoskeletal and Skin Diseases
(NIH): http://www.nih.gov/niams/
NLM—National Library of Medicine: http://www.nlm.nih.gov/
medlineplus/arthritis.html
The Arthritis Society (Canada): http://www.arthritis.ca/home.html
U.S. Food and Drug Administration: http://www.fda.gov/

Copper Jewelry and Arthritis 261
A Final Word on Questionable Health Claims
An intensive sweep of the Internet in 1998 targeted over 1200 sites making
potentially false or deceptive advertising claims regarding the prevention,
treatment, or cure of arthritis, cancer, diabetes, heart disease, AIDS, and
multiple sclerosis. The ‘‘Health Claim Surf Day’’ spotlighted the tremendous
power of the Internet as an information resource for consumers. It also
uncovered the Web’s dark side: ‘‘it also provides promoters of fraudulent
health products and treatments easy access to consumers from all over the
world’’ (92).
REFERENCES
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11
Role of Copper in Anti-inflammatory
Therapy and the Potential for Its
Transdermal Application
Jurij J. Hosty´nek
Department of Dermatology, University of California at San Francisco
School of Medicine, San Francisco, California, U.S.A.
Roberto Milanino
Facoltà di Medicina e Chirurgia, Sezione di Farmacologia,
Dipartimento di Medicina e Salute Pubblica, Università di Verona,
Verona, Italy
INTRODUCTION
Copper is an essential trace element (ETE), critical for a variety of biological
processes, for example hemoglobin synthesis, enzyme activation, particularly
that of superoxide dismutase, and, more generally, as a key component of
mitochondrial, cytoplasmic, and nuclear enzyme systems. Absorbed mainly
from dietary sources (organ meats, shellfish, nuts, whole-grain cereals),
copper absorption and utilization is interdependent with that of other metals,
including zinc, iron, cadmium, and molybdenum. Significant stores of copper
are found in liver, muscle, and bone. It is required for bone formation,
cardiac function, keratinization, and tissue pigmentation, where it binds to
proteins, preferentially sulfur- and nitrogen-containing ligands. In mammals,
ceruloplasmin (CP), a multifunctional endogenous plasma glycoprotein, is
responsible for copper transport in the blood where it may bind some 60%
to 70% of the metal present. Physiological factors that require involvement
267

268 Hosty´nek and Milanino
of copper as a remedial factor induce CP production in the liver, thus
assuring an adequate transport medium in response to increased translocation
requirements (1). Total plasma copper is a reliable indicator of
circulating CP, as a highly significant correlation exists between the two
values (r ¼ 0.8), in rats as well as humans (2,3).
Copper plays an active role in inflammation, and animal models show
that the anti-inflammatory activity of the metal is amplified when complexed
with nonsteroidal anti-inflammatory drugs (NSAIDs). As a rule, such complexes
also become more effective and less irritant than the parent ligands (4–9).
It has been hypothesized that supplementation with exogenous copper
may bring remedial action in inflammatory disease such as arthritis (A), and
it was widely demonstrated that intravenous (IV) and subcutaneous (SC)
administration of copper solutions is effective in controlling edema induced
in a variety of animal screens (6,10–12).
The inflammatory condition comprises a wide spectrum of diseases that
include rheumatoid arthritis (RA), childhood arthritis, carpal tunnel syndrome,
osteoarthritis, lupus, and gout—conditions associated with pain,
stiffness, and swelling in and around the joints. It affects one out of every
three adults and is the most common cause of disability in the United States.
It results in 75,000 hospitalizations and 39 million outpatient visits annually
(13). The majority of those affected by inflammatory diseases, of all
racial and ethnic groups, are less than 65 years of age, as onset is most often
seen between the ages of 25 and 50 years. RA and osteoarthritis, the most
common forms of A, are characterized by chronic joint inflammation eroding
cartilage and bone. Some forms of A, such as RA, psoriatic arthritis, and
lupus, can affect multiple organs and cause widespread symptoms. RA is
characterized by nonspecific, usually symmetric inflammation of the peripheral
joints, and may lead to destruction of articular and periarticular
structures. It is a chronic syndrome generally classified as an autoimmune
disease, and a genetic predisposition has been identified. Control of the condition
in humans is achieved through use of immunosuppressive drugs, and
symptoms can be reduced by treatment with anti-inflammatory compounds.
The majority of those afflicted improve symptomatically with conservative
treatment in the early stages of the disease, but many are severely disabled
over the course of their lives, as a cure is not known (14,15).
TRADITIONAL AND MODERN THERAPIES FOR RA AND
RELATED DISORDERS
Copper Metal—Bracelets and Rings
Historically, use of copper in the form of the metal, as verdigris (basic cupric
acetate) or vitriol (cupric sulfate), has been a time-honored practice since
antiquity, recognized for its anti-inflammatory activity (16). The potential

Copper in Anti-inflammatory Therapy 269
for its activity as an anti-inflammatory agent by transdermal delivery,
however, remains subject to controversy because scientific studies designed
to demonstrate therapeutic benefits for arthritic conditions through dermal
contact with metallic copper or its compounds have been inadequate. Quantitative
data for percutaneous penetration of the metal’s putative oxidation
products, which may be generated in contact with skin in humans, are not
available. Because no beneficial effect could be statistically documented
for skin contact with copper devices, their use was and still is relegated to
‘‘quack’’ medicine. It is unfortunate that this term has been used when
the term ‘‘not understood’’ would have been more appropriate.
Persisting lay use of copper devices to the present day and assertions of
beneficial effects obtained seem to warrant an independent study of the
metal to objectively evaluate its effectiveness, separating activity from placebo
effect, even if the current state of anti-inflammatory medicine casts a
doubt over the economic value of such studies. A first step in reinstating
the metal in the armamentarium of anti-inflammatory remedies would be
a quantitative, controlled study of copper release from the metal brought
in intimate contact with human skin in vivo.
Release of Copper in Contact with the Skin
In order to confirm the long-standing assertions that skin contact with copper
bangles has an AI effect due to skin penetration of solubilized metal,
Walker and Keats performed experiments to verify that copper metal was
oxidized and dissolves in artificial sweat. In samples where initial copper
concentration was of the order of 2 10 5 M, after 24 hours the samples
turned blue and the concentration had increased to 2 10 3 M Cu. The
same authors also measured the weight loss from copper bracelets due to
corrosion worn by volunteers. Bracelets measuring 22 1.3 0.1 cm lost
an average of 13 mg per month (17).
Weight losses of 0.1% to 0.8% were observed again in copper bracelets
weighing 14 g used in a trial involving volunteers and lasting one month (18).
Such variation can be explained by differential mechanical wear. An alternative
explanation is the variability in subjective sweat composition and sweating rate.
Skin Penetration by Copper Compounds
The only experiment that allows calculation of a diffusion constant Kp for
a copper compound was performed by Walker. Bis(Glycinato) copper (II)
complex, a copper compound likely to be formed with skin exudates, was
applied to cat skin in vitro (19). A radioactive ( 64 Cu) 0.05 M solution in
physiological saline applied to excised cat skin resulted in a steady-state
transport rate Kp ¼ 24 10 4 cm/hr. All other diffusivity data for copper
compounds had to be deduced from clinical observations or physical analytical
methods on animals and humans following exposure.

270 Hosty´nek and Milanino
In vivo application of copper oleate in a lanolin-petroleum base over
24 hours to human back skin resulted in a significant increase in urinary copper
levels over several days (20).
Following topical application of lipophilic Cu(II) salicylate and -phenylbutazone
complexes, designed to study their penetration and biodistribution
in rats, 64 Cu was eliminated almost quantitatively, primarily in feces, an indication
that Cu(II) rapidly permeates the dermal barrier (21).
Electron microscopy of skin treated topically with copper acetate led
to the observation that copper initially localizes in the intercellular spaces;
subsequently, the cell membranes of viable cells are penetrated as well, and
the metal accumulates in and around the cell nucleus (22).
By electron probe analysis and analytical electron microscopy, occurrence
and levels of copper were traced across human cadaver skin following
application of CuSO4 using Franz cells. Copper was observed to enter the
outer sc cells at high concentrations; it then encounters an apparent barrier
approximately halfway within the epidermis, falling below the detection limit
(0.1% by weight), to reappear at the granular interface. There, it was primarily
seen traversing an intercellular route through the granular layer, to reach
the spinosum layer (Warner, personal communication).
Scope and Limitations of the Quantitative Structure Activity
Relationships Approach to Diffusivity of Metal Compounds
Principles that govern diffusion of exogenous compounds through biological
membranes such as the skin are prejudicial towards penetration by metal
complexes in general, governed as they are by numerous factors difficult to
evaluate a priori.
Mathematically derived (mechanistic) models predictive of percutaneous
penetration have been developed based on in vitro and in vivo data
through regression analysis of experimental results, and serve to predict diffusivity
of untested structures. Within limits of molecular parameters such
as size, lipid solubility, or hydrogen bonding, these models apply to organic
compounds such as drugs, pesticides, cosmetic ingredients, etc., and can predict
their dermal absorption with increasing accuracy (23–26). The elements
required for construction of such predictive models can be reduced to the
criteria of structure and physical properties (27). No such reduction is feasible
when modeling diffusion of electrolytes, however. Attempts to model
the dermal absorption process of metal compounds, and those of transition
metals in particular, have been thwarted owing to a number of confounding
factors, as became evident from in vivo and in vitro experiments. Because
movement through biological membranes is highly element and chemical
species specific, molecular physicochemical parameters alone do not suffice
to model their migration into and through the strata of the skin. A number
of factors are closely inter-related and their combined effects are neither
entirely understood nor predictable. Unless the dynamics of in situ changes

Copper in Anti-inflammatory Therapy 271
in valence (oxidation state), or electrophilic reactivity, among others, can be
factored in, the diffusivity of metallic elements eludes modeling. Furthermore,
the limited experimental data available so far from in vitro and in vivo
experimentation for metal compounds have been acquired under disparate
conditions and are too scarce considering the number of metals and
metalloids of variable valence existing as free ions or forming chelates, coordination
compounds, or complexes with electron donors such as oxygen,
sulfur, or phosphorus, abundant in biological systems. The resulting database
is too limited for the development of predictive algorithms. Computer
calculations have been used to predict skin diffusivity for chemical structures,
and of conventional AI agents in particular (28–30), but not for cupriphores.
DRUG THERAPY
Aspirin, Salicylates, and Other Anti-inflammatory Drugs
Aspirin, first used as an antipyretic, has also been recognized for its analgesic
activity, and it was elaborated in a plethora of derivatives (salicylates). Relatively
safe, these derivatives, as well as aspirin itself, continue in use as the
cornerstone of AI drug therapy to the present day. Introduced to replace cortisone,
an AI hormonal agent first isolated in the 1930s, they act on the swelling,
heat, and pain of inflammation that is suffered by A victims. They are relatively
safe and inexpensive. Given PO they can induce GI symptoms affecting the
gastric mucosa, however, and erosion and bleeding gastric ulcers may result.
Besides derivatives of salicylic acid, NSAIDs from other chemical classes
have been evaluated for their anti-inflammatory activity: N-arylanthranilic
acids, e.g., mefenamic acid, derivatives of arylacetic acid, e.g., ibuprofen,
diclofenac, piroxicam, aniline and p-aminophenol derivatives, acetaminophen,
etc. (31). Given PO, their dosage is still limited, however, as they also carry
the risk of GI symptoms.
Recent anti-inflammatories are selective NSAIDs, such as the family of
COX-2 cyclo-oxygenase inhibitors Vioxx (rofecoxib), Bextra (valdecoxib),
or Celebrex (celecoxib). In contrast to aspirin, however, they do not impair
platelet adhesiveness, which may lead to cardiovascular disorders, and in
addition are suspected to induce psoriasis (32).
Gold
Systemic treatment with soluble gold (I) salts (chrysotherapy) represents a
continuum in the armamentarium of rheumatologists since the beginning
of drug therapy to combat arthritis, as it affords rapid symptomatic relief.
The different aspects of gold therapy, positive and negative, are therefore
reviewed here in greater depth. Initially used for the treatment of tuberculosis
until the 1930s, promoted by Forestier chrysotherapy found its principal therapeutic
application in the treatment of RA (33). Use continues in addition to

272 Hosty´nek and Milanino
salicylates or other NSAIDs if the latter do not bring sufficient relief or
suppress active joint inflammation. A serious limiting factor in systemic application
of soluble gold salts has been the high incidence of adverse reactions,
as some degree of toxicity appears inevitable at effective dose levels. The most
common toxic effects involve the skin and mucous membranes, including
pruritus, allergic contact dermatitis (ACD), and stomatitis, but gold compounds
can also be toxic to the hematopoietic organs and the kidney. Gold
therapy must be discontinued when any of those manifestations appear,
which occurs in up to one-third of patients (34,35).
Parenteral preparations include gold sodium thiomalate, gold thioglucose,
or gold thiosulfate, and are administered IM at weekly intervals: 10 mg
the first week, 25 mg the second, and 50 mg/wk thereafter until a total of 1 g
has been given or significant improvement is apparent. When maximum
improvement is achieved, dosage is decreased to 50 mg every 2–4 weeks (14).
In active RA, chrysotherapy provides rapid pain relief, and can induce partial
or complete remission in as high as 80% of cases, as long as therapeutic regime
is maintained. Even with sustained therapy, however, there is roentgenological
evidence of progression of the disease. Eventually, all patients relapse in 3–6
months when therapy is discontinued; the condition then returns to the pretherapeutic
status and the disease continues on its natural course (36).
Despite the high incidence of serious adverse effects, chrysotherapy is
still widely accepted, since for many forms of arthritis, given parenterally,
orally, or more recently, intra-articularly, gold (I) salts still represent the
most effective therapy (37).
Thanks to its anti-inflammatory properties, over the past half century
gold has found a number of applications, such as treatment of juvenile and
adult rheumatoid arthritis via intramuscular injection of the water-soluble
thiomalate, thioglucose, or thiosulfate (38). Other new uses include the treatment
of such skin diseases as pemphigus and psoriatic arthritis, which
involve an immunological component (39). Such use is no longer practiced
in the United States. Following parenteral therapy, which involves relatively
high doses, gold was seen to persist in the organism for several months.
Carried by plasma, bound to alpha-globulin, it is carried to practically every
tissue, including the skin where it becomes evident in characteristic blue-gray
discoloration, or chrysiasis, most often appearing on the face and hands,
areas exposed to sunlight, hair, and nails (36,38,40).
While for nearly three-quarters of a century gold salts have been used to
relieve the symptoms of arthritis, just how the metal exerts its antiarthritic
effect is not clear, as it does not have a known biological function itself.
Stoyanov and Brown (41) propose an indirect role by gold since they find that
it specifically binds to a copper-activating protein, thus turning on production
of a protein pump involved in copper transport, mobilizing endogenous
copper reserves. The biological effect of gold antiarthritic drugs may thereby
consist in their effects on copper management in eukaryotic systems.

Copper in Anti-inflammatory Therapy 273
Immune Reactions in Chrysotherapy
Due to increasing systemic exposure of patients for therapeutic purposes
over the past 50 years, gold is now recognized as a significant factor in the
etiology of cellular and humoral immunity (39,42). It is also an inducer of
circulating immune complexes (43,44).
Systemically, gold compounds have modulatory effects on immediate
and delayed immune responses, both in vitro and in vivo (42), causing type I
hypersensitivity (contact urticaria syndrome), cell-mediated (type IV) sensitization
or ACD, as well as type III or Arthus reactions involving antigen–
antibody complexes. Hypersensitivity reactions to both the monovalent
and trivalent forms of the metal have been described. While ACD is not
uncommon due to the widely practiced systemic therapy using gold
compounds, or the intimate skin contact with metallic gold, urticarial reactions
are extremely rare. The relatively high incidence of delayed skin
reactions has been noted since the introduction of gold compounds for
the treatment of rheumatoid arthritis, and is seen in more than 50% of
patients so treated (45,46). Although not clinically relevant to the most part,
the rate of sensitization to gold seems to be on a steady increase, with comparable
prevalence in the United States, Europe, and Japan since routine
screening for gold allergy was started in those countries (47–51). Injection
of water-soluble gold complexes is associated with a high risk of sensitization
in the form of a type I hypersensitivity response (52). Repeated
exposure to gold salts in sensitized persons does not necessarily lead to
recurrence of dermatitis, however. This indicates a gradual buildup of tolerance
(53). Abnormally high serum IgE levels together with eosinophilia have
been associated with gold therapy, but remit on cessation of such therapy
(54,55). Nephrotic syndrome, and glomerulonephritis in particular, is noted
as a consequence of parenteral or oral administration of gold in its organic
form. Certain gold compounds, such as Ridaura (auranofin), given orally in
which gold is complexed to trialkylphosphines, are suspected of giving rise
to circulating immune complexes (type III hypersensitivity) (42). As an electrophile
reacting with native protein, the metal forms a complete antigen
that induces specific antibody formation, which, in turn, gives rise to the glomerular
injury observed (43,44,56,57). In patients treated parenterally with a
variety of gold compounds, the metal is seen to be retained in significant
amounts (over 80% of the amount injected over 39 days of administration)
bound to the plasma component alpha-globulin as a gold protein complex.
The amounts excreted (primarily in urine) were not related quantitatively to
the amounts injected (38), and retention may increase the risk of immune
complex glomerulonephritis in patients receiving gold preparations.
In a large Swedish study involving 823 dermatology patients, the incidence
of 8.6% positive reactions to gold was recorded, without any signs of
an irritant etiology (58). Recent confirmation of this result now makes gold

274 Hosty´nek and Milanino
the second most common contact allergen (in the Swedish population) after
nickel. Thus, routine patch testing of dermatitis patients in Europe has
revealed a rate of positive readings approximating 10% and 14.3% in North
America (59,60). Based on test results by the North American Contact
Dermatitis Group, with 9.5% positive, gold is the sixth most common
allergen among dermatology patients in the United States (61). A similar
prevalence is noted in Europe and Japan (50,51).
Systemically provoked flare-ups of contact allergy to gold appear as
another characteristic of response to that allergen. Intramuscular injection
of gold sodium thiomalate was reported to reactivate previously involved
but clinically healed skin. It also led to corresponding pathobiochemical
changes in the venous blood of the patients (i.e., activation of neutrophil
granulocytes and release of several cytokines). Similarly, intradermal test
sites that had healed 9 to 24 months previously were reactivated (62–64).
The diverse skin reactions do not appear to depend on concentrations
of the metal in the skin. Patients receiving cumulative doses of 1 to 50 g of
gold (I) salts may not experience adverse reactions (65), and there is no consensus
on optimal or limit in dosage. Thus, tolerance for gold salts is highly
individual, and patients require constant monitoring for toxic manifestations.
In summary, while contact of intact skin with metallic gold is not
known to induce hypersensitivity, its occurrence in context with systemic
exposure through injection of soluble salts for therapeutic purposes is
now considered to be an unavoidable side effect in the risk–benefit balance
of anti-inflammatory therapy.
Corticosteroids
Corticosteroids are effective oral and intra-articular anti-inflammatories, but
their efficacy diminishes with time and they do not prevent joint destruction.
Their use is also associated with various side effects, and steroid therapy is
only recommended on failure of less hazardous drugs (14).
Superoxide Dismutase
Anti-inflammatory copper-dependent metalloenzymes, superoxide dismutase,
have been developed for the treatment of arthritic diseases (66). Two
such preparations, Ontosein and Orgotein, have been shown to be safe
and effective for the treatment of established rheumatoid and osteoarthritis
when given IV or IA into knee and hip joints in single or multiple doses.
They cannot be dosed PO, however, due to gastric inactivation.
Immunosuppressants
Methotrexate, cyclosporine, and others are used for severe RA, but bring
the risk of major side effects: liver disease, bone marrow suppression, and
potentially, malignancy (14).

Copper in Anti-inflammatory Therapy 275
The search for new anti-inflammatory agents has had limited success
so far, owing to the lack of insight into the causes and mechanisms of
inflammatory diseases.
Physiotherapy
Other than surgical intervention, relief from arthritic pain was sought in a
number of noninvasive approaches: ultrasound, thermotherapy, balneotherapy,
electrotherapy, laser therapy, acupuncture, and even tai chi.
PRECEDENTS IN TOPICAL DELIVERY OF
ANTI-INFLAMMATORY AGENTS
Topical anti-inflammatory formulations have a history of successful application.
An early precedent for topical treatment of rheumatic discomfort were
thermogenic ointments based on methyl salicylate (wintergreen) and menthol,
at present marketed as Ben-Gay by Pfizer. Also some of the drugs mentioned
above, such as NSAIDs, corticosteroids, immunosuppressives, and also
dimethylsulfoxide and capsaicin were reported to be clinically effective in
placebo-controlled trials (67). The argument for developing copper
complexes with proven analgesics can be made invoking the findings by
Sorenson and others, cited elsewhere, that copper–ligand chelates show
anti-inflammatory activity superior to that of the ligands themselves.
ROLE OF COPPER IN AI ACTIVITY
Knowledge sent down from early historic times has been combined with
modern pharmacology. Recognition of the essential and therapeutic role
that endogenous copper plays in the control of inflammatory processes
led to the concept of introducing the ETE into state-of-the-art pharmacology,
thus combining the two elements. A hypothesis first: the idea of using
exogenous copper in combination with analgesics and anti-inflammatories
may turn out to be a successful concept with augmenting effects in modulation
and control of inflammatory processes.
Inflammation is tissues’ response to injury and represents the first step
of the repair process towards normalization. While many anti-inflammatory
agents may reduce some aspects of inflammation, they do not necessarily
involve repair processes towards correcting the cause of the disease. Copper
complexes, on the other hand, were demonstrated to promote tissue repair
processes, and they involve activity in a number of diseases with inflammatory
components other than arthritis. Their therapeutic use includes infection,
cancer, diabetes, and epilepsy (7,8). Copper as an ETE is present in the
mammalian organism only in minimal concentration as free ionic copper
(10 18 mol, with a range between 10 11 and 10 19 mol) and is predominantly
complexed with proteins and amino acids, ligands that are subject to dynamic

276 Hosty´nek and Milanino
exchange in response to physiological requirements and homeostatic equilibria
(8,68). In animals and humans, copper levels change in response to
inflammation.
Heilmeyer and Stuwe (69) had first observed that serum copper rose
with arthritic disease, and fell again on remission. Stores of copper normally
immobilized are bound to metallothionein, released, and bound to CP that
binds over 60% of total serum copper. CP was shown to possess remarkable
anti-inflammatory characteristics (70) in vivo. Experimental induction of
either acute or chronic inflammation in the rat promotes accumulation of
copper in some body compartments that are known to be directly or indirectly
involved in the development (and remission) of the inflammatory
processes (2). Thus, inflammation appears to determine an increased
demand for copper by the organism, a requirement fulfilled by adjusting
the intestinal absorption without depleting the major body stores (11,71).
A copper-supplemented diet exhibits an antiarthritic action, and, conversely,
copper deficiency appears associated with proinflammatory effects, as
observed in animals on a copper-deficient diet (3).
In the 1960s and 1970s, publications reported on the anti-inflammatory
activity in animal models for copper compounds in different forms: as
mineral salts, as enzymes, or complexes, when given IM, intraperitoneal,
SC, and, most of all IV. It was also suggested that active copper complexes
were formed within the organism, as their activity in animal models of
inflammation was found to be greater than either treatment with inorganic
copper salts or the complexing agents alone, while the parent compounds or
ligands were inactive in test models of inflammation even at larger screening
doses, in coordination with copper they were found to be active (4,5). Thus,
the therapeutic activity of a ligand appears to depend on its association with
(endogenous or exogenous) copper. This may be attributed to the ligand acting
as the carrier for the metal, or copper in the complex acting as a catalyst,
interfering with the inflammatory process.
As reviewed by Bonta, occasional publications reported that
sodium-3(N-allylcuprothiouredo)-l-benzoate and cuprous iodide had antiinflammatory
activity and that Cu(II)(salicylate)2 had fever-lowering effects
in various animal models of inflammation or fever. In addition, copper
complexes of antiarthritic drugs, including salicylic acid, acetylsalicylic acid,
penicillamine, and several corticoids, were found to be more active than the
parent drugs. A comparison of copper, gold, and silver thiomalate and
thiosulfate complexes in models of inflammation revealed that the copper
complexes were effective, while the gold and silver complexes were virtually
inactive. As a general rule, copper complexes are less toxic and suppress the
ulcerogenic effect of many of the ligand drugs themselves. Those early
reports of anti-inflammatory activity of copper complexed with various
ligands were based on IV or parenteral administration (72).

Copper in Anti-inflammatory Therapy 277
Based on this hypothesis, the compounds investigated subsequently
included coppercomplexes of well-known antiarthritic drugs, including steroidal
and nonsteroidal anti-inflammatory drugs: niflumic acid, D-penicillamine,
hydrocortisone, dexamethasone, dimethylsulfoxide, clopirac, ketoprofen,
(þ)-naproxen, indomethacin, mefenamic acid, diclofenac, ibuprofen among
a number of others. All the copper complexes of those drugs have been
found to be active or more effective anti-inflammatory agents than the parent
drug (8,73).
Oral and local administration of Cu(I) and Cu(II) complexes in animals
mostly led to equivalent responses in the edema model. Since the two
forms of the metal undergo different reactions in vitro, a catalytic function
is attributed to exogenous copper ion, also suggesting that both
Cu(I) and Cu(II) act by the same anti-inflammatory mechanism involving
a common metabolite (74). The ETE, normally absorbed as Cu(II) ion,
is carried in the plasma by CP, albumin, transcuprein or low molecular
weight complexes such as those with different amino acids in that oxidation
state, since, as is the case for Fe(II), Cu(I) can potentially prime the
harmful Fenton reaction. As in the cell interior, the metal seems to exist
almost exclusively in the Cu(I) form, the necessary reduction step is performed
by plasma membrane reductase, an enzyme likely to be expressed
by all body cell types. The cells themselves are well equipped with copper
chaperones for the control of intrinsic Cu(I) toxicity. These metallochaperone
proteins escort copper ions directly to enzymes that require
the metal to function, such as the antioxidant copper–zinc superoxide
dismutase.
In a review of anti-inflammatory activity of exogenous copper,
Milanino et al. (11) also concluded that copper indeed is active as an acute
anti-inflammatory agent irrespective of chemical form given, including inorganic
copper salts.
Administration of exogenous copper is effective in controlling inflammation
associated with arthritis, and may lead to complete (albeit temporary)
remission. Sorenson (8) reviewed the AI activity of several dozen copper complexes,
mostly exploratory and several as commercial drugs, the categories of
ligands including non-anti-inflammatory compounds such as amino acids,
heterocyclic carboxylic acids and amines, among others. The AI activities
reported were based on oral or parenteral administration. However, copper
salts compounded with salicylic acid or ethyl salicylate have also been shown
to have AI activity in animal models of inflammation following topical
application (75).
Another indication of the anti-inflammatory action of copper is the rise
of serum copper in pregnancy, in tandem with remission of RA, which is
attributed to the protective AI action due to an increase in CP levels. This
is followed by a postpartum relapse to pre-existing RA (76).

278 Hosty´nek and Milanino
PAST USE OF COPPER CHELATES IN THE TREATMENT
OF RHEUMATOID ARTHRITIS
Mixtures of copper salts and salicylic acid or ethyl salicylate in aqueous
or glycerol-dimethyl sulfoxide solutions were tested successfully for antiinflammatory
activity following topical application in a variety of animal
models of inflammation. During the period 1940–1950, the copper-containing
compounds sodium 3-(N-allylcuprothiouredo)benzoate (Cupralene) and
Alcuprin, given IV, sodium meta-(N-allylcuprothiocarbamide), given IM,
and Cu(II) (4,6diethylammonium-8-hydroxyquinoline)2 sulfonate (Dicuprene
or Cuprimyl), given IM, were reported by Forestier to be effective in
treating a variety of arthritic diseases, including acute and chronic RA,
chronic polyarticular gout, ankylosing spondylitis, and disseminated spondylitis.
Forestier et al. (77) provided evidence in support of the suggestion that
these copper complexes were superior to gold therapy.
Work linking copper and inflammation has been done by Sorenson
(4,5), who demonstrated that copper (II) acetate alone has an AI activity
in experimental models of inflammation, with the suggestion that the active
metabolites for AI activity are copper chelates. Sorenson and Hangarter (6)
reported on clinical data obtained for some 1500 patients suffering from diffuse
connective tissue disease over 30 years, noting that toxic side effects
such as those occurring with chrysotherapy were not observed with copper
therapy. Although these results were confirmed by others, the advent of
hydrocortisone and the belief that it was going to ‘‘cure’’ arthritis led to the
disuse of these preparations.
Hangarter’s preparation of aq. copper salicylate, an aqueous mixture of
12.5 mM of sodium salicylate and 0.04 mM Cu(II) chloride (Permalon), given
IV, was more effective than the other clinically used complexes in the treatment
of A and other degenerative diseases. That complex was effective in treating
acute rheumatic fever, acute and chronic RA, erythema nodosum, and sciatica
either with or without lower back spinal degenerative disease. From a total of
620 patients treated over a 20-year period, 65% benefited by remission through
this therapy, with a mean remission time of three years, 23% experienced
improvements in their condition, while 12% remained unchanged (7). Hangarter
mentions a lack of gastrointestinal complaints in the cohort of patients,
which were common when only sodium salicylate was used in therapy. In
1970, the manufacture of Permalon was discontinued for economic reasons,
however, and Hangarter’s approach to therapy came to an end.
TRANSDERMAL DELIVERY OF ANTI-INFLAMMATORY COPPER
CHELATES VS. CONVENTIONAL (SYSTEMIC)
ANTI-INFLAMMATORY THERAPY
Since it appears important to maintain a needed minimal level of copper in
tissues for the control of inflammation (78), the skin may be an adequate site

Copper in Anti-inflammatory Therapy 279
for the delivery of cupriphores at maintenance levels. The case can therefore
be made in favor of dermatologicals for copper-based therapy in the treatment
of inflammation, since it may avoid patient discomfort and potential
disadvantages such as the first-pass metabolism or idiosyncratic drug reactions
and GI side effects inherent in conventional routes of administration
(PO, ID, IP, IV, IA) (79–81). Due to lessened systemic clearance, local tissue
accumulation of the AI agent would be possible in the treatment of conditions
such as muscle pain and inflammation by low-level, controlled, and
sustained release. As computer simulation of metal–ligand equilibria reveals
that salicylates and similar complexes are thermodynamically unstable in
plasma (82,83), this implies that the exogenous form of copper administered
is different from the complex formed with endogenous ligands, and has no
direct bearing on the anti-inflammatory activity. It thus appears fortuitous
that most AI activity of copper has been studied using salicylate derivatives,
in animals and humans, as AI activity of copper was exhibited by a wide variety
of other ligands also. It is the increased availability of endogenous copper
per se, which affords protection against inflammation. This is particularly
encouraging in the search for skin-diffusible copper compounds, since the
mechanism by which copper acts as the actual anti-inflammatory and antiarthritic
agent, and what its bioactive moieties are, is still under discussion.
In topical therapy, substances can be applied in concentrations that
would be too toxic for systemic exposure. Given orally, compounds may
contribute to gastric irritation (e.g., the salicylates), and the pharmacologically
active form of copper complexes is also likely to be compromised (84).
Copper complexes dissociate at the varying pH prevailing in the GI tract
and become subject to sequestration and biliary excretion. Diffusion
through the skin on the other hand avoids irritation in the GI tract and
first-pass inactivation by the liver, and avoids discontinuity in therapy due
to lack of patient compliance.
Several theoretical and practical obstacles need to be overcome. With
data presently available, progress in the development of copper-based
dermatologicals for inflammatory disease therapy is severely limited. One
key element awaiting development is data on the penetration of the ETE
through human skin in any of its forms: as a polar mineral salt, or as a
low molecular weight hydrophilic or larger lipophilic complex.
Although dermatological copper preparations are, or were available,
e.g., the topical anti-inflammatory copper-salicylate gels Alcusal 1 or Dermcusal
1 , found to be effective in animals, they are not commonly employed
for the treatment of RA in humans. This can be attributed to skepticism on
the part of the medical profession due to the folkloristic component attached
to such therapy, and also due to a lack of adequate controlled human studies.
New treatments must be shown to be effective through unbiased, objective
testing before the medical community acknowledges its potential benefits.
Thus, an independent randomized placebo-controlled trial of the copper

280 Hosty´nek and Milanino
salicylate–ethanol complex failed to show the effectiveness of the product:
applied to the forearm it was no better than the placebo gel for (subjectively
evaluated) pain relief in patients with osteoarthritis of the hip or knee (85).
Skepticism prevails vis-à-vis copper-based therapy because its mode of
anti-inflammatory action in the organism is insufficiently understood, and
stability and fate of complexes delivered is unknown. For the electrophilic
copper ion in particular, primarily binding sites in proteins may interfere
with the dermal diffusion process.
A major effort should be dedicated to the cutaneous delivery of cupriphores
in quantities significant for local therapeutical activity. A first step is
a de minimis approach: certain types of compounds could be defined as
priority candidates for topical application of cupriphores for which Kps
can be determined using standard in vitro permeation protocols, in order
to put them on a relative scale of diffusivity. Such priority would go to small
molecular weight complexes with ready solubility in polar media, which do
not exhibit skin toxicity, bound to ligands of established safety. A prime
such category that comes to mind are:
Reaction products with low molecular weight essential amino acids
such as glycine or histidine. Molecular size is a key determinant for
optimal flux across the skin, and low molecular weight complexes will
also determine the ability of plasma copper to diffuse into tissues.
Unstable copper complexes with toxicologically safe profiles,
which may release the metal to endogenous protein-binding sites
in significant amounts, such as citrate, gluconate, histidinate, or
sebacate. The latter category may also enhance the amount of copper
absorbed, overall thanks to significant SC diffusion.
The case can be made in favor of dermatologicals for copper-based
therapy in the treatment of localized musculoskeletal disorders, as it allows
targeted application of higher local drug concentrations too toxic systemically,
avoiding or minimizing patient discomfort and untoward effects
potentially incurred using conventional forms (IM, PO, IP, IV, IA) of treatment.
Targeted exposure focusing on specific areas of inflammation becomes
possible, providing higher local tissue concentration, rather than the blunderbuss
approach through systemic therapy, which renders the agent subject
to the vagaries of enterohepatic circulation and biliary excretion (81).
As an instance, given orally compounds may contribute to gastric irritation
or worse (e.g., the salicylates), and the pharmacological activity of
copper complexes is also likely to be compromised (84). The compounds dissociate
at the varying pH prevailing in the GI tract, and become subject to
sequestration and excretion.
Also, when a localized condition such as arthritis is treated systemically,
the drug reaches the intended target only after resorption, passage
through the liver, and distribution throughout the entire system. Apart from

Copper in Anti-inflammatory Therapy 281
the potential for selective accumulation in the target area upon systemic dosing,
the quantity of drug, which eventually becomes available on the intended
target, is thus limited. Following systemic application, the pharmacokinetics
are entirely different from those resulting from topical exposure. In the latter
case, the drug can be applied directly targeting the afflicted part of the
anatomy, i.e., the sites of clinical significance, with the potential for local
skin reaction as the worst case. Since dissipation of the therapeutic agent
due to transport throughout the system does not occur, topical, local therapy
affords higher tissue concentrations, especially in the outer skin layers, which
would be too toxic systemically.
The potential for copper activity as an anti-inflammatory agent by
transdermal delivery, however, is subject to controversy. The studies designed
to demonstrate therapeutic benefits of topical therapy for arthritic conditions
with either metallic copper or dermaceuticals such as Alcusal or Dermcusal 1
by Walker et al. were not conducted lege artis. They were based on subjective
patient assessment, obscured by placebo effect. Also, quantitative data for
percutaneous penetration of copper’s putative oxidation products generated
in contact of the metal with skin in humans, a critical factor in accrediting
beneficial effects of copper bangles, have not been determined. Walker measured
such absorption, albeit by one degree of separation, by gravimetrically
determining the amount of copper lost in function of contact time only, and
evaluating the resulting beneficial effect in afflicted patients by using questionnaires
and psychologic parameters (18).
Shackel et al. assessed the efficacy and safety of a copper-salicylate gel
in osteoarthritis patients in a randomized, double-blind, placebo-controlled
study, to be sure. But they were again based on self-assessment of pain,
scores decreasing in both the treated and placebo groups, with no significant
difference between the two groups for decrease in pain (85).
Aside from the copper bangles issue, one missing, important factor for
a convincing case of copper’s potential benefits is thus the deficiency in
adequate, systematic research into the benefits of topical application of cupriphores.
A measure of copper penetration through human skin in any of its
forms, as polar mineral salts or as the more lipophilic chelates, is missing.
Subcutaneous Administration of Cupriphores
Favorable for transdermal therapy is evidence of anti-inflammatory activity
obtained when bypassing the epidermal barrier through sc injection of
cupriphores. Transdermal therapy as an effective route was documented
repeatedly in animal models by sc administration of anti-inflammatory copper
compounds (74,86,87). Notably, one of those investigations concluded
that sc administration of some copper complexes was more effective than if
they were given orally, while noting also that no difference was observed
between the activities of copper (I) and copper (II) compounds in those

282 Hosty´nek and Milanino
experiments (74). The latter serves to counter the argument that, in applying
copper compounds, speciation of that ETE may be of importance.
That levels of copper introduced into plasma strongly correlate with
the anti-inflammatory effect was demonstrated when a wide range of
copper–ligand complexes, administered sc, i.e., reaching the plasma without
having to traverse the skin, were effective in controlling edema induced in
animal screens (4,5,88).
Positive such results were also obtained through sc administration of
copper complexes by Lewis et al. An AI effect by sc dosing of copper aspirinate
and other copper compounds in different animal species has been
described (86).
Precedents in Effective Transdermal Cupriphore Delivery
in Animals and Humans
High levels of 64 Cu radioactivity localized in loci of inflammation following
dermal application of copper chelates by Beveridge indicated that exogenous
copper was sequestered at inflammatory sites, and provides evidence
of percutaneously absorbed copper complexes (75).
Two lipophilic formulations of Cu(II) salicylate, Alcusal 1 and
Dermcusal 1 , were developed by Walker et al. for dermal treatment of
inflammatory conditions. Alcusal 1 is a copper salicylate–ethanol complex
prepared by refluxing Cu(II) hydroxide with salicylic acid in anhydrous
ethanol, Dermcusal 1 from copper salicylate prepared in a dimethyl sulfoxide–
glycerol solution. Applied on the shaved dorsal skin of rats, they significantly
reduced artificially induced paw edema and suppressed experimental arthritis,
thus demonstrating facile cutaneous penetration (21,89,90).
When the copper salicylate–ethanol complex was applied topically on
the skin of human volunteers, analysis of elevated plasma salicylate levels
did indicate that penetration had occurred. However, upon application on
self-diagnosed arthritis and rheumatism patients, upon subjective evaluation
no statistically significant results in pain relief could be documented (18).
Overall, organic copper compounds to treat RA, albeit promising in
the hands of many clinicians over time, led to inconsistent, and sometimes
contradictory results. Pharmaceutical copper preparations are available
but are not widely employed for the treatment of RA due to the effective
and continuing use of chrysotherapy and the application of steroidal preparations,
and also due to skepticism on the part of the medical profession
in consideration of the folkloristic component attached to therapy based on
copper. Further research proved inconclusive because of deficient experimental
design in selection and evaluation/classification of patient cohorts,
in the diagnostic criteria applied, the lack of controls, the confounding placebo
effect, and to subjective evaluation by patients themselves, which was
based on perception. Adding to such procedural deficiencies came the lack

Copper in Anti-inflammatory Therapy 283
of marketing incentives at the pharmaceutical industry level. Thus, use of
copper derivatives to treat RA and related inflammatory diseases gave
way to other emerging anti-inflammatories such as Cox-2 inhibitors, which
inhibit inducible cyclo-oxygenase enzymes and thereby inhibit prostaglandins,
important mediators of inflammation. Health complications have
become associated with that class of drugs now: aggravation of pre-existing
ulcers, increase in the incidence of thrombosis, rise in blood pressure among
others (32,91,92). They thus give rise to criticism and prompt inquiries on
the part of health officials, favoring efforts to find alternatives for the treatment
of inflammation.
Should the epidermal barrier prove too formidable for diffusion of
copper chelates to reach concentrations required for clinically meaningful
analgesic and anti-inflammatory effect, however, several alternative techniques
are now are available to carry drugs into or through the skin.
Percutaneous Drug Delivery Systems and Methods
Chemical Adjuvants
There are limitations in the selection of drugs that may qualify for passive
transdermal delivery (93). Passive percutaneous penetration, whether transcellular,
via intercellular lipids or shunts, depends on several limiting factors,
including molecular weight, optimally below 500 Da, or volume, lipophilicity,
charge, and drug potency. To overcome some of the obstacles to passage
of drugs through the SC, the first and most restricting barrier of the skin,
research has endeavored to find chemical adjuvants, which would overcome
the relatively low SC permeability to enhance penetration. Enhancement to
deliver therapeutic levels has been achieved with formulations in which the
active ingredients were compounded with terpene derivatives, e.g., linalool,
limonene, menthone, or eugenol (94,95). Enhancers leading to higher drug
levels in the dermis have chemical characteristics, which have the potential
of disrupting the arrangement of the intercellular lipid bilayers and liquefing
lipid sheets, structural changes that paradoxically are the very prerequisites
for enhanced diffusion. Because exogenous penetration enhancers can
exhibit overt toxic or irritant effects, their continued use for improved drug
delivery is put in doubt.
Dermal absorption of copper complexes by simple passive diffusion, particularly
of those with higher molecular weight, e.g., Cu(II)-diclofenac or
Cu(II)-ibuprofenato complex with AI effect, may be problematic (96,97).
Methods of entrapment (liposomes) or active delivery by physical methods
have been developed, however, and could also be successfully applied.
Liposomes
Liposomes (LSs) have been shown to efficiently deliver the large molecular
weight class of proteins, such as antigens for immunization studies (98).

284 Hosty´nek and Milanino
Depending on the molecular moiety to be delivered, LSs can be formulated
to incorporate a wide array of substances as a payload, either in the water or
in the lipid compartments, to contain antibodies, large molecular weight
ligands, or polysaccharides.
LSs are spherical uni- or multilamellar lipid/water vesicles with diameters
in the micron-to-nanometer range. They are formed when thin lipid
films or lipid cakes of natural, nontoxic phospholipids and cholesterol are
hydrated and stacks of liquid crystalline bilayers become fluid and swell.
The hydrated lipid sheets detach during agitation and self-assembly to form
large, multilamellar vesicles that prevents interaction of water with the hydrocarbon
core of the bilayer at the edges. The size of the particles can be reduced
by sonication or extrusion. Properties of lipid formulations can vary depending
on the composition (cationic, anionic, or neutral species) and degree of
fatty acid saturation; the same preparation method can be used for all lipid
vesicles regardless of composition (99).
Biological particulate materials can be incorporated in LSs without
compromising their structure or activity by exposing them to organic
solvents, or light-sensitive compounds can be protected from radiation by
incorporating a light-absorbing compound in the liposomal structure. Also,
sensitive pharmaceuticals or labile bioactive materials can be encapsulated
in an LS designed for controlled or sustained release, to degrade only at a
target-specific application site. The LS structure is very similar, physiologically,
to the material of cell membranes. When a formulation containing
liposomes is applied to the skin, for example, the LSs are deposited on
the skin and begin to merge with the cellular membranes. In the process, the
LSs release their payload of active materials into the cells. As a consequence,
not only is delivery of the actives very specific—directly into the intended
cells—but the delivery takes place over a longer period of time. Thus, LSs
as a delivery system can be designed for slow or fast release of both a hydrophilic
or hydrophobic payload (100).
The SC is recognized as the main barrier to skin penetration, as well as
the major pathway for intracellular and intercellular drug delivery. Going a
step further, Scheuplein (101–104) suggested that hair follicles also act as
shunts, permitting rapid transport of charged and large polar molecules.
Feldmann and Maibach (105) observed increased percutaneous transport
through skin areas with high follicular density, also suggestive of follicular
delivery. Refinements in methods of observation later confirmed this route
for a wide range of molecules, particularly identifying particulate moieties
such as LSs, localizing them in follicular and sebaceous areas to act as drug
reservoirs (106,107). The potential for targeted percutaneous delivery of
copper complexes of differing polarity and large molecular weight in LSs
as carriers to the hair follicle may also offer an alternative in achieving therapeutically
effective levels of that anti-inflammatory agent. Particularly
attractive appears liposomal delivery of copper complexed with conventional

Copper in Anti-inflammatory Therapy 285
anti-inflammatories that exhibit an augmenting activity, such as ibuprofen or
diclofenac (96,97). These complexes appear unsuitable for conventional
passive diffusion due to molecular size and limited solubility. Potentially
nontoxic, degradable, and nonimmunogenic (108,109) LS acting as slow
release vehicles could thus create a copper reservoir in the SC.
Physical Methods
Enhancement in skin absorption can be achieved by occlusion or different
forms of energy input, characteristic of active transfer of drugs through the
skin. These approaches utilize patch techniques, iontophoresis, sonophoresis,
or electroporation to overcome the severe restrictions imposed on passage
through the skin.
Patches
Transdermal absorption is a slow process of passive diffusion, driven by the
gradient between the given concentration in the external delivery system and
the zero concentration present in the skin. The delivery system using patches
keeps the penetrant in continuous intimate contact with the skin with occlusion
over days. Given adequate diffusion rates, steady blood levels can
thereby be achieved through such continuous and even delivery over
extended periods, which is preferable to the spikes and troughs occurring
when administration occurs by other routes such as PO or IV. As an
instance in delivery of analgesics, the fentanyl patch acts for 72 hours,
providing long-lasting pain relief. Some other major drugs currently qualified
for marketing as transdermal patches, intended for daily or weekly
application, are clonidine (hypertension), estradiol (estrogen deficiency),
estrogen-progestin (contraception), fentanyl (analgesic), lidocaine (anesthetic),
nicotine (smoking cessation), nitroglycerine (angina pectoris), scopolamine
(motion sickness), and testosterone.
Iontophoresis
Iontophoresis facilitates diffusion of ionic drugs and higher molecular
weight compounds such as peptides. By anodal or cathodal electrophoresis
negatively or positively charged drugs can be delivered respectively through
the patient’s skin; under application of electric current one electrode serves
to deliver the drug, the other to close the circuit. By this method luteinizing
hormone-releasing hormone, a decapeptide of 1200 Da molecular weight
was successfully dosed (110).
Electroporation
Electroporation is a technique that delivers high voltage pulses to the
skin, causing transient changes in cell membranes or lipid bilayers (111).

286 Hosty´nek and Milanino
Such pretreatment of the skin can facilitate delivery of large, polar molecules
such as peptides.
Sonophoresis
Low-frequency ultrasound treatment of the skin, or sonophoresis, has been
developed as another physical method to promote diffusion; this is for
highly hydrophilic drugs like mannitol (112).
CONCLUSIONS
Copper as an essential trace element and its role in current anti-inflammatory
therapies are reviewed, from the controversial, age-old practice of wearing
copper bangles to the copper complexes as anti-inflammatory drugs. Oxidation
products formed by copper in contact with the skin and their potential
for their skin diffusion are discussed. Considering the potentially negative
side effects as well as the discomfort associated with anti-inflammatory
therapies using current methods of application, transdermal delivery of
cupriphores appears to be an attractive alternative worth exploring, although
the present lack of data on the skin diffusivity for all categories of copper
compounds is a shortcoming, which must first be overcome. Should transdermal
delivery of copper chelates lessen or suppress inflammation, untoward
systemic effects associated with conventional modes of anti-inflammatory
therapy could be lessened or avoided.
Aside from general principles of percutaneous absorption and prerequisites
for skin penetration by chemicals, the potential for topical delivery
of copper complexes is reviewed with the intent to impart renewed impetus
to the evaluation of transdermal anti-inflammatory therapy with cupriphores.
Strategies are identified for their passive or active diffusion in order
to penetrate the stratum corneum barrier, with the purpose to compensate
with exogenous sources delivered through the skin potential, local shortfalls
in endogenous reserves of copper.
Evidence gathered in animals and humans affected by inflammatory
processes suggests that percutaneous administration of copper may represent
an appealing therapeutic approach, particularly in consideration of
the unfavorable aspects of adverse side effects and discomfort associated
with more conventional therapies. Also, applied dermally, the drug in the
target tissues can be expected to exceed the concentrations achievable
through more conventional, systemic avenues.
At this time, several theoretical and practical obstacles prejudice
endeavors in the development of copper-based dermatologicals
for the therapy of inflammatory diseases, regardless of the form
in which that metal may be applied.

Copper in Anti-inflammatory Therapy 287
Although dermatological copper preparations are available, they
are not commonly employed for the treatment of RA in humans.
Skepticism on the part of the medical profession persists due to a
lack of controlled efficacy studies.
A missing building block towards making a convincing case for the
potential benefits in transdermal therapy is the lack of quantitative
data on the penetration rate of copper through human skin, in any
of its forms: derivatives of skin-identical amino acids, prepared in
analogy with peptides potentially formed in contact of the metal
with the skin (glycinate, histidinate, etc.), as polar mineral salts,
or as the more lipophilic chelates (e.g., as ibuprofen or diclofenac
complexes) applied as liposomes.
Principles governing the transfer of metal compounds through biological
membranes, including the skin, are prejudiced by numerous
factors that are difficult to anticipate a priori. For copper in particular
as an ETE, binding sites in proteins may interfere with the
dermal diffusion process, resulting in degradation or blockage
(homeostatic controls).
Studies designed to demonstrate significant therapeutic benefits for
arthritic conditions through skin contact with metallic copper or its
complexes in humans, e.g., with oxidation products that may be
generated in contact of the metal with the skin have not been conducted
in a convincing manner.
That transdermal AI therapy can be effective has already been demonstrated
in animal models by administration of a number of copper
compounds (74,86,87). The investigation by Brown et al. (74) concluded that
administration of some of the complexes was more effective than when given
orally, and no obvious differences were observed between the activities of
copper (I) and copper (II) compounds.
Endeavors should focus on achieving transport of copper compounds
through the skin in pharmacologically significant quantity. To overcome
some of the obstacles, as a first step in a de minimis approach, small
molecular weight amino acids could be investigated for skin diffusivity.
Such copper chelates may release the metal to protein binding sites in significant
amounts.
Technical arguments elaborated at this time in favor of transdermal
cupriphores indicate that:
1. Regardless of the ligand with which copper reaches the organism,
the metal in the initial complex recombines with endogenous proteins
and is homeostatically directed to the target locus in the
organism where the metal exercises its function.
2. Both Cu(I) and Cu(II) in the exogenous complex are equally
effective in reducing inflammation, speaking for a common

288 Hosty´nek and Milanino
(recombinant) transition state of that highly reactive metal within
the organism, a characteristic shared by other transition metals.
Copper exerts its role as ETE, thanks to that ability of the outer
electrons in the 3d and 4s orbital shells, which allow it to fluctuate
between the monovalent and the divalent oxidation state. Such
facile changes in valence play a decisive role for their transfer
across cell membranes and epithelia, including the skin.
OUTLOOK
Summing up the factors enumerated, the essential groundwork for the transdermal
delivery of cupriphores appears to have been laid. An array of
technologies for their delivery are available; preliminary results obtained
so far in animals and humans warrant pursuit of that form of therapy
and make a resurgence in the use of copper as anti-inflammatory possible.
Albeit dermal diffusion of copper complexes formed by the metal in contact
with the skin have been demonstrated (not published), transdermal application
in form of patches would appear to be a more efficient method of
dosing exogenous copper.
Closer definition of copper complex stability is not necessary, since
homeostasis takes control of the ETE in whatever form exogenous copper
reaches the organism and converts it to endogenous complexes appropriate
for anti-inflammatory function. Should cupriphores be adapted as
patches for needle-free transdermal delivery, by use of chemical adjuvants,
physical preconditioning of the skin, or imbedded in LS, some of the traditional
remedies for RA (e.g., gold therapy or copper bracelets) may be
replaced by effective and less controversial approaches. Transdermal therapy
would bring with it the advantages of lesser immunotoxicity, of selfadministration,
avoidance of discomfort, and economy.
ABBREVIATIONS
A arthritis
AI anti-inflammatory
CP ceruloplasmin
ETE essential trace element
GI gastrointestinal
IA intra-articular
IM intramuscular
IV intravenous
L ligand
LS liposomes
MT metallothionein
NSAIDs nonsteroidal anti-inflammatory drugs

Copper in Anti-inflammatory Therapy 289
PO per os
QSAR quantitative structure activity relationships
RA rheumatoid arthritis
sc subcutaneous
SC stratum corneum
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Absorption, 22
degree of, 76
dermal, 22
measuring percutaneous, 45
limitations and problems
in, 46
radioactivity method for, 48
in vitro method for, 45–47, 52
in vivo methods for, 47–52
of metal compounds, 22, 46
skin, 22
Acute inflammation, 162, 163, 166
Acute periodontal disease, 161
Adjuvants, chemical, 283
Alcusal 1 , 200
Allergic contact dermatitis (ACD),
116, 272
copper, 128
test concentrations for, 123
diagnostic tool for, 120
predictive test for, 126
See also Allergy, delayed-type
Allergy
delayed-type, 116, 120, 139
immediate-type, 119
Alloys
brass, 5
bronze, 5–6
cast, 4
Index
295
[Alloys]
chromium coppers, 5
compositions of, 104
copper, wrought, 4
copper–nickel, 2, 6
nickel–silver, 6
solubility of particulate, 107–108
Alpha-ceruloplasmin, 74
Aluminum bronzes, 5
applications of, 6
characteristics of, 6
American Conference of Governmental
and Industrial Hygienists
(ACGIH), 98
Amino acids, 11
Analgesic–narcotic principles, 152
Analytical electron microscopy, 69
Angry back syndrome, 131
Anti-inflammatory (AI)
activity, 68, 167
copper chelates as, 243
topical delivery of, 275
Anti-inflammatory drugs,
nonsteroidal, 238
Anti-inflammatory potentials, 150
Anti-inflammatory properties, 150
Antiarthritic
agents, 162
application, 244

296 Index
[Antiarthritic]
copper potential, 159
drugs, 162
Antirheumatic agents, 187
Antiseptic properties, 151
Area under the curve (AUC), of
triplicate stripping
experiments, 88
Arthritic deformation, 240
Arthritis
analgesic products, 255
pain relief, 249
patients with copper jewelry, 82
remedy for, 238
rheumatoid, 238, 250
treatment, 243
Artificial sweat
copper ion in, 13
skin penetration data for, 13
Aspirin, 190, 271
for analgesic activity, 271
an antipyretic, 271
Atomic absorption analysis, 14
Biliary excretion, 279
Blue copper oxidases, 186
Bracelets, 240
copper, 239
therapeutic, 240
weight loss, 247–248
Brass, (red or yellow), 5
Breathable tape, 82
Bronze
alloy, 5–6
aluminum, 5
silicon, 5
tin, properties of, 5
Bronze Age, 5
Carrageenan-induced paw edema
(CPE), 163, 165
Carrageenan-induced pleurisy
(CP), 165
Cast alloys, 4
Ceruloplasmin, 164, 167
concentrations, 172
[Ceruloplasmin]
in serum, 181
as markers, 172
transcription of, 171
Chemical adjuvants, 283
Chemical irritants, 98
Chlorophyllin copper complex, 102
Cholinergic urticaria reaction, 139
Chondrocytes, sensitivity of, 207
Chromatography, ion exchange, 11
Chromium copper alloys, applications
for, 5
Chronic inflammation, 162,
168–170, 171
Chronic inflammatory agents, 152, 162
Chronic inflammatory disease, 238
Chronic inflammatory processes, 163
Chronic systemic pathologies, 199
Chrysotherapy, 272
applications of, 282
immune reactions in, 273
Complementary and alternative
medicine (CAM), 246
Connective tissue metabolism, 206
Contact urticaria syndrome, 116
Copper
absorption, 67, 74, 72, 83, 244, 267
cutaneous, 67, 74
dermal, 255, 270, 283
administration
exogenous effects, 184–203
percutaneous, 199–203
routes of, 184–186
subcutaneous and intraperitoneal,
188–191
allergy, 121, 123, 128
alloy
applications of, 2
biofouling resistance of, 3
castability of, 3
characteristics of, 2–3
corrosion resistance of, 2
fabricability of, 3
families, 3
friction rates of, 3
wear rates of, 3
ANA 68 alloy, 104
animal models for, 276

Index 297
[Copper]
anti-inflammatory activity of, 67, 188,
203–216, 268, 275
anti-inflammatory effects, 91–92
antimicrobials, 102
as applications, 84–85
articles, corrosion of, 68
baseline values of, 82–84, 88
blood clotting factors, 8
bracelet, 239, 255, 269
myth, study of, 249
weight loss, 13, 269
chelates in rheumatoid arthritis
(RA), 278
chemicals, 3
chlorophyllin, 102
comparison of nickel and, 87, 89
complexes, 7–8, 158
compounds, 67
concentration, 172, 181, 182
content of hair and skin, 89
contraceptive effects, 14
corrosion
in environment, 8
in physiologic media, 9
cross-reactivity between palladium
and, 133
decontamination, 84–85
deficiency, 101, 163, 255
alimentary regimen, 181
experimental animals study, 163–170
feeding, 163
organism, 75, 76
dental alloys, released from, 100
in dental materials, 124
depletion, proinflammatory effect
of, 163
deprivation, 163
derivatives, used as pesticides, 118
dermal irritation by, 111
in dermatology, 118
dermatopharmacokinetics, 75
detection of limits for, 81–82
diffusible compounds, 118
diffusion, 67, 69, 81, 83, 269, 288
diffusivity, 269, 270
electron configuration and reactivity
of, 8
[Copper]
electron microscopy for, 270
electron probe analysis for, 270
electrophilic reactivity of, 271
endogenous, 102
epicutaneous tests for, 135
exogenous, 103, 277
effects of, 91
exposure, 97
types, 125
formation of, 99, 255
and hemolytic anemia, 101
in human organism, 14
human stratum corneum penetration
by, 81
hypersensitivity, 115, 132
immune reactions
case reports, 138
individual reports, 138
reports of, 115–116
immune response, 125
as immunogen, 117
immunogenic potential of, 123
immunologic contact urticaria
due to, 127
in intrauterine devices (IUDs), 100,
115, 125
intravenous administration
of, 186–188
ionic, 243
irritation
epidemiology of, 100
incidence of, 100
in skin and mucosa, 103
in jewelry, 82, 98
machinability of, 3
mechanical properties of, 3
properties of, 2–4
thermal and electrical conductivity
of, 3
types of, 6
wrought, 3
leaching of, 10
LG-1/ANA 68, 105
LM-hard gold alloy, 104
in mammalian organism, 275
in medicine, 243
application, 244

298 Index
[Copper]
development of, 240
metabolism disorder, 101
metal
dissolution, 82
solubilization, 98
in metallic bonding, 8
metallurgy of, 117
micro/ANA 68, 104
midi/ANA 68, 105
noxious effects on, 91
occlusion, 84–85
oleate, in vivo application of, 118
oral administration of, 191–198
oxidases, blue, 186
oxidation
in body fluids, 117
states for, 8
oxidation potential of nickel and, 90
parameters for, 270
patch test material, 133
penetration
under occlusion, 87–88
under semiocclusion, 88–89
PGE2 released, 109–110
pharmaceutical, 282
pharmacological activity of, 280
pharmacology of, 155
physiological factors for, 267
plasma, 268
polystyrene, 104
population-based studies, 134–140
powder, 84
predictive immunology test
results for, 119
properties of, 1–2, 83, 240
protein, multifunctional, 158
quantitative data for, 102
radioactivity of, 282
removal by sequential tape stripping,
86–87
salicylate gel, 281
salicylate–ethanol complex, 282
scope and limitations of, 270
sensitivity, 126
sensitization potential of, 115
as sensitizer, 117
skin contact with, 83, 269
[Copper]
skin penetration by, 84, 86, 269
skin reactions to, 82
skin-diffusible, 279
sources of exposure to, 9
status
in blood and urine, 170–173
in solid tissues and inflamed areas,
173–179
stems, 97
stripping, 84–85
in synthetic sweat, 99
systemic allergic contact dermatitis
due to, 129–130
systemic lupus erythematosus disease
activity in, 254
T-cell-mediated reactions to, 123
as therapeutic agent, 238
therapeutic uses of, 240
in therapy, 150
threshold limit values applicable to, 98
toxicity, 72
and therapeutic indices of, 91–92
toxicological considerations of, 91
transdermal anti-inflammatory action
of, 102–103
transdermal therapy by, 278, 279
urticaria, screening procedure, 131
utilization, 267
in vitro assays, 109
in vivo assays
agarose overlay test in, 106
comparison of the different
tests in, 108
erythrocyte lysis assay of, 107
implantation test of, 103–105
toxicity test using murine
macrophages in, 107
Copper metabolism, studies on
endogenous, in acute chronic
inflammation, 170–179, 179–184
Copper sulfate
diffusion of, 72
pharmacological use of, 153
Copper-dependent enzymes, 186, 211
lysyl oxidase, 199
role of, 206
Copper–nickel alloys, 6

Index 299
Copper/zinc bracelets, study of, 249
Corneocytes, 23
Corpus hippocraticum, 154
Corrosion-resistant materials, 3
Corticosteroids, 274
CP. See Carrageenan-induced pleurisy
CPE. See Carrageenan-induced paw
edema
Cranial trepanation procedure, 153
Cupric ion, testing of, 119
Cupriphores
effective transdermal, 282
subcutaneous administration of, 281
Cutaneous treatment, efficacy of, 199
Cyclo-oxygenase (COX)
activities, 205
pathways, 205–206
Cytochrome-c oxidase activity, 183
Cytolysis, degree of, 106
Delayed contact stomatitis, 124
Dental alloy contact dermatitis, 124
Dental materials
copper alloys used in, 124
copper sensitivity induced by, 126
Dermal absorption experiments,
22–27, 75
approaches for, 25
descriptors of, 25
percent of, 26
permeability coefficient of, 25–26
Dermatitis
dental alloy contact, 124
systemic contact, 117
Dermatome, use of, 44
Dermatopharmacokinetics, 26
Dermatosis, 116
Dermatotoxicology, 26
Dermcusal 1 , 200
Dermis, 22
Diagnostic equivocation, 133
Diet, semiquantitative assessment of, 248
Dietary copper deficiency, effects of, 168
Diffusion
characteristics of, 76
in copper, 269, 270
experiments, in vitro, 76
[Diffusion]
Fick’s law of, 25
Kalia’s equation, 23
membrane analysis, 76
passive, 75
rates of, 68
Dimethyl acetamide, 37
Dimethyl formamide, 37
Dimethyl mercury, 22
Dimethylsulfoxide (DMSO), 37
applications of, 122
skin penetration by, 122
as vehicle, 122
Divalent oxidation state, 68
D-penicillamine, 162
Drug
delivery systems, 76
intercellular, 284
percutaneous, 283
ionic, 285
therapy, 271
Elastic connective tissue, 24
Electric resistance, 29
Electrolyte
composition, 10
concentration, 11
Electromotive forces and potential, 10
Electron probe analysis, 69
Electron-dense granules, 24
Electrophilic chromic ion [Cr(III)], 39
Electrophilic metals, 31
Electroporation, 286
Electropositivity, 8
Endogenous origin of skin exudates, 33
Energy dispersion X-ray (EDAX)
analysis, disadvantage
of, 105–106
Enterohepatic circulation, 280
Enzyme, 209
lipolytic, 12
as marker, 183
Epidermis, constitution of, 22, 23
Epithelial cells, 23, 69
Erythrocytes, copper concentration, 181
Erythropoiesis, 203
Escherichia coli, 3

300 Index
Essential trace element (ETE), 67
for biological processes, 294
elimination of, 22
Excited skin syndrome, 133
Exogenous copper administration,
effects of, 184–203
Extracellular-matrix
macromolecules, 199
Femur fracture, 174
Fertility-related phenomenon, 15
Fibroblast–keratinocyte cocultures, 109
Fick’s law of diffusion, 25
Fluid fraction, 166
Food and Drug Administration
(FDA), 242
Gastric irritation, 279
Generic therapeutic remedies, 152
GHK-Cu. See Glycyl-L-histidyl-Llysine-Cu(II)
Glomerulonephritis, immune complex
of, 273
Glycyl-L-histidyl-L-lysine-Cu(II)
(GHK-Cu), 198, 199, 206
Gold, 271
antiarthritic drugs, biological effect
of, 272
ring study, 250
therapy
anti-inflammatory properties
for, 272
soluble gold salts for, 272
therapeutic application, 271
Gold sodium thiomalate, 272
intramuscular injection of, 274
pathobiochemical effects of, 274
Gold sulfhydryl compounds, 239
Gold thioglucose, 272
Gold thiosulfate, 272
contact allergy to, 274
modulatory effects on, 273
tolerance for, 274
GPMT
on 20 guinea pigs, 119
as predictor of skin
sensitization, 119
Gyrotory TM water bath model, 85
HaCaT cells, cytotoxic potential
in, 110–111
Hair follicles, 33
Hapten–protein conjugate, 120
Haruspecism, definition of, 152
Hematochemical serum markers, 163
Hemoglobin, synthesis of, 157
Histamine
activity, 203–205
release, 203–205
Histopathology, 105
Homeostasis, 42, 74
Homeostatic mechanisms, 74, 76
Human
abdominal epidermis, 72
inflammatory process, 171
plasma or serum, 118
rheumatoid arthritis, 158, 184
serum albumin (HAS), 120
skeleton, 156
skin
dermatoglyphics, 83
skin features, 12
stratum corneum penetration by
copper, 81
metal analysis of, 85
statistical analysis of, 85
Hypercupremia, 172
Hypersensitivity
asymptomatic contact, 124
copper, 115
copper allergic, 121
population studies of, 132
diagnostic tests for, 119
immediate allergic, 120
open test for, 119
radioallergosorbent test (RAST)
for, 120
ICAMs. See Intercellular adhesion
molecules
ICU. See Immunologic contact
urticaria
IgE antibodies, 120
production of, 124
Immune system development and
reactivity, 212–216

Index 301
Immunologic contact stomatitis, 124
Immunologic contact urticaria (ICU),
115–116, 119
due to copper, 127
See also Allergy, immediate-type
Immunosuppressants, 274
In vivo methods, 47–51
advantages of, 49
approaches for, 48
conclusions of, 50
semiquantitative, 50
skin stripping in, 50
Inductively coupled plasma-mass
spectrometry, 69
Inductively coupled plasma–mass
spectroscopy (ICP-MS)
analysis, 85
Inflammatory arthropathies, 252
Inflammatory reaction, 165, 207
Intercellular adhesion molecules
(ICAMs), 211, 212
Intrathoracic injection, 165
Intrauterine devices (IUDs), 98
Ion, cupric, testing of, 119
Ion exchange chromatography, 11
Ionic copper, release of, 243
Ionic drugs, 285
Iontophoresis, 285
Irritant contact dermatitis (ICD),
incidence of, 101
Irritant chemicals, 98
Kalia’s diffusion equation, 23
Keratinocytes, 23
Lactic dehydrogenase (LDH), 107
Leukocyte
activity, 207–212
migration, 166, 207–212
Lipid bilayers, intercellular, 283
Lipolytic enzymes, 12
Lipophilic compounds, 28
Lipophilic formulations, 282
properties of, 284
Liposomes (LS), 284
biological particulate materials
in, 284
oxidation, 76
particle size of, 284
Local lymph node assay (LLNA),
cuprous chloride in, 119
Mammalian sweat, 11
Membrane transport, 8
Membrane-bounded organelles, 24
Menkes’ syndrome, 101–102
Metabolism, of connective tissue, 206
Metal(s)
corrosion rates between two, 90
oxidation of, 89
Metal detection, 53–56
analytical methods for, 53
atomic absorption spectrophotometry
(AAS) for, 54–55
electron spin resonance (ESR)
for, 55
inductively coupled plasma–atomic
emission spectroscopy
(ICP-AES) technique for, 53
inductively coupled plasma–mass
spectroscopy (ICP-MS)
technique for, 50, 53–54
particle-induced X-ray emission
for, 56
stable-isotope inductively coupled
plasma–mass spectroscopy
technique for, 54
Metal surfaces, sebum and sweat in, 36
Metal–ligand equilibria, computer
simulation of, 279
Metallo-element–deficient animals, 163
Metallothionein, 32
Micronutrients, physiological dynamics
of, 75
Microparticle-induced X-ray emission
(PIXE), 29
Mononuclear leukocytes, activity
of, 207
Musculoskeletal disorders, localized, 280
Mycobacterium butyricum, 168

302 Index
Nephrotic syndrome, 273
Neurotoxic antimicrobial
hexachlorophene (1–3), 22
Nickel
cross-reactivity between copper
and, 133
cross-reactivity between palladium
and, 133
dermatopharmacokinetics of, 50
epicutaneous tests for, 135
ion-specific T-cell clones, 133
reactivity of, 133
Nickel ion–specific T-cell clones, types of
reactivity of, 133
Nickel–silver, 2
alloys, 6
Nitric oxide synthase activity, regulation
of, 206
Nonarthritic copper deficiency, 170
Nonsteroidal anti-inflammatory drugs
(NSAIDs), 187, 188, 191, 200,
238, 268
piroxicam, 210
NSAIDs. See Nonsteroidal antiinflammatory
drugs
Optimal flux, 280
Orbital shells, 68
Osteoarthritis, 249
Patch testing
copper sulfate under, 121
for delayed-type allergy, 120–121
detection of allergens by, 121
diagnostic, 37
optimal solvent in suspected
delayed-type copper allergy,
123, 126
positive reactions, 90
role of vehicle in, 121–122
Periodontal disease, 158
acute, 161
Perkin-Elmer 4300 dual view ICP optical
emission spectrometer, 85
Permeability coefficients, measurement
of, 72
Permeation protocols, in vitro, 280
Petrolatum, 122–123
Pharmacokinetics, 281
principles, 47
Pharmacological therapy, 159
Phorbol miristate acetate
(PMA), 209
Physiotherapy, 275
Pilosebaceous glands, 12
Placebo effect, 247
Plasma copper concentration, 172
Plasma glycoprotein, multifunctional
endogenous, 267
Plasma-membrane barrier, 68
PMNLs. See Polymorphonuclear
(neutrophils) and mononuclear
leukocytes
Polymorphonuclear (neutrophils),
activity of, 207
Polymorphonuclear (neutrophils) and
mononuclear leukocytes
(PMNLs), 207, 209
migration, 210–211
phagocytosis, 207
phagosomes, 208
respiratory burst, 209
Polypropylene tape, 85
Proinflammatory effect, 163
Prostaglandin E2 (PGE2), 109
Prostaglandin-synthetase activity, 169
Provocative use test (PUT), 126
Pseudoscience, survey of, 243
Quantitative cutaneous diffusion
rates, 67
Quantitative structure–activity
relationships, 28
RA. See Rheumatoid arthritis
Radioactivity, 26
Radioallergosorbent test (RAST)
inhibition test, 120

Index 303
Reactive oxygen species (ROS)–induced
reactions, 209
Red blood cell, copper level, 181
Repeat open application test
(ROAT), 126
Rheumatic patients, 243
articular erosion in, 250
Rheumatoid arthritis (RA), 76, 171,
179–184, 268
autoimmune disease, 268
chronic syndrome, 268
copper chelates in, 278
inflammatory disease, 268
patients, 161
spontaneous remission of, 162
symmetric inflammation of, 268
therapy of, 72
traditional and modern therapies
for, 268
treatment of, 238, 250
ROS. See Reactive oxygen species–
induced reactions
‘‘Rusters’’, 11, 34
Sebaceous glands, 12
Sebum, classes of, 12, 36
Serum copper levels, determination
of, 167
Silicon bronzes, 5
Skin
absorption, 22
by different sites of the
body, 42
methods for measuring
percutaneous, 45
afflictions, 243
age of, 42
allergic reactions in, 39
arsenic in, 45
aspects of metals, 23
chemical microenvironment on, 9
chromium applied on, 44
compounds formed by metals in
contact with, 31–35
diffusion of
metal compounds, 35
[Skin]
nonelectrolytes, 28
paths of, 28
discoloration of, 44
electrolytes across, 29–31
principal conditions of, 29
electron micrographs of, 14
function as diffusion barrier, 23
gold in contact with, 90
immune reactions to chemicals
in, 116
as membrane, 22
metabolism (red/ox), 44
nickel
as powder on, 50
in contact with, 90
organic compounds, 28–29
penetration, 21
by DMSO, 122
by nickel chloride, 43
by xenobiotics, 22, 33
permeant categories of, 28
pH effect on, 39
reactions to copper, 82–83
sensitization, 119
structure of, 23
Surface, formation of free acids
on, 99
sweat and sebum, 9, 90
in vitro, in function of polarity, 38
Skin-diffusible derivatives, 90
Skin-diffusible oxidation products, 32
Skin diffusion
endogenous factors for, 41
age of skin as, 42
homeostatic control mechanisms,
43–43
regional variation, 41
exogenous factors for, 35
counterion and molecular
volume, 37
dose, 35–36
nature of chemical bond, 38
solubility, 39
time postapplication, 41
valence, 39
vehicle as, 36–37

304 Index
[Skin diffusion]
polarity determinates for, 38
variables determining, 35
Skin diffusivity rates, 67
Skin prick device, sterile, 120
Skin prick test (SPT), 119
for immediate-type allergy, 119–120
positives, 120
Solubility, in polar media, 280
Sonophoresis, 286
Sorenson’s hypothesis, soundness
of, 191
Staphylococcus aureus, methicillinresistant,
3
Steady-state transport rate, 14
Steroid therapy, 274
Stomatitis
delayed contact, 124
immunologic contact, 124
systemic anaphylactic, 124
Stratum corneum (SC), 23, 98
absorption of copper into, 91
barrier, 286
components of, 24
copper diffusion through, 81,
83, 89
depots in, 90, 40–41
intercellular lipid domains in, 30
occlusion of, 84
protein reactivity, 40–41
stripping, 83
swelling of basal, 116, 122
Student t-test, 85
Superoxide dismutase, 243
anti-inflammatory copper-dependent
metalloenzymes, 274
Superoxide metabolism, regulation
of, 168
Sweat
components, 10, 33
duct, 29
glands, 33
mammalian, 11
mean body, 11
mean levels of, 33
proteins and amino acids in, 34
Sweating rate, 12
Syndrome
contact urticaria, 116
nephrotic, 273
Systemic anaphylactic stomatitis, 124
Systemic contact dermatitis, 117,
129–130
Talcum powder, formulation of, 22
Therapeutic agent, dissipation of, 281
Therapeutic properties of copper, 150
Therapy
drug, 271
effectiveness of topical, 249
gold, 271
pharmacological, 159
steroid, 274
Thermal stimulation, 12
Thermogenic ointments, 275
Threshold limit values (TLV), 98
Throat inflammation, 155
Tin bronzes, 5
Topical therapy, 279
Total serum copper, increase of, 171
Transdermal
absorption, 285
applications, 288
Transepidermal water loss
(TEWL), 23
Transition metals
in biological systems, 8
characteristic for, 8
Transpore TM tape, 85
Tuberculosis and syphilis, infection
of, 157
Ulcerogenic effect, 276
VAP-1. See Vascular adhesion protein-1
Vascular adhesion molecule-1
(VCAM-1), 211
Vascular adhesion protein-1 (VAP-1),
211, 212
VCAM-1. See Vascular adhesion
molecule-1

Index 305
Wilson’s disease (WD), 101, 162
Wilson’s disease protein (WND), 185
Wounds, treatment of
postoperative, 151
Wrought alloys, 4
Wrought copper alloys, 3
Xenobiotics, Fick’s law applied
to, 22, 25
Zinc absorption, measurement
of, 75