The Antioxidant Network of the Stratum corneum

Abstract

Many studies have demonstrated beneficial health effects of topical antioxidant application; however, the underlying mechanisms are not well understood. To better understand the protective mechanism of oxogenous anti-oxidants, it is important to clarify the physiological distribution, activity and regulation of antioxidants. Also, the generation of ROS by the resident and transient microbial flora and their interaction with cutaneous antioxidants appears to be of relevance for the redox properties of skin. Our studies have demonstrated that alpha-tocopherol is, relative to the respective levels in the epidermis, the major antioxidant in the human SC, that alpha-tocopherol depletion is a very early and sensitive biomarker of environmentally induced oxidation and that a physiological mechanism exists to transport alpha-tocopherol to the skin surface via sebaceous gland secretion. Furthermore, there is conclusive evidence that the introduction of carbonyl groups into human SC keratins is inducible by oxidants and that the levels of protein oxidation increase towards outer SC layers. The demonstration of specific redox gradients within the human SC may contribute to a better understanding of the complex biochemical processes of keratinization and desquamation. Taken together, the presented data suggest that, under conditions of environmentally challenged skin or during prooxidative dermatological treatment, topical and/or systemic application of antioxidants could support physiological mechanisms to maintain or restore a healthy skin barrier. Growing experimental evidence should lead to the development of more powerful pharmaceutical and cosmetic strategies involving antioxidant formulations to prevent UV-induced carcinogenesis and photoaging as well as to modulate desquamatory skin disorders.

Full text

............................
Oxidants and Antioxidants in Cutaneous Biology
..

............................
Current Problems in
Dermatology
Vol. 29
Series Editor G. Burg,Zu
¨
rich

............................
Oxidants and Antioxidants in
Cutaneous Biology
Volume Editors Jens Thiele, Jena
Peter Elsner, Jena
36 figures, and 5 tables, 2001

............................
Current Problems in Dermatology
Library of Congress Cataloging-in-Publication Data
Oxidants and antioxidants in cutaneous biology / volume editors, Jens Thiele, Peter Elsner.
p.; cm. – (Current problems in dermatology; vol. 29)
Includes bibliographical references and index.
ISBN 3805571321 (hard cover : alk. paper)
1. Skin – Pathophysiology. 2. Free radicals (Chemistry) – Pathophysiology. 3.
Antioxidants. 4. Active oxygen – Physiological effect. I. Thiele, Jens. II. Elsner, Peter,
1955– III. Series.
[DNLM: 1. Oxidants – adverse effects. 2. Skin Diseases – physiopathology. 3.
Antioxidants – therapeutic use. 4. DNA Damage. 5. Oxidative Stress. 6. Signal
Transduction. 7. Skin – radiation effects. 8. Ultraviolet Rays – adverse effects. WR 140
O973 2000]
RL96.O947 2000
616.507–dc21
00–048668
Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents
Ô
and
Index Medicus.
Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and
dosage set forth in this text are in accord with current recommendations and practice at the time of publication.
However, in view of ongoing research, changes in government regulations, and the constant flow of information
relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for
any change in indications and dosage and for added warnings and precautions. This is particularly important
when the recommended agent is a new and/or infrequently employed drug.
All rights reserved. No part of this publication may be translated into other languages, reproduced or
utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying,
or by any information storage and retrieval system, without permission in writing from the publisher.
Ó Copyright 2001 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland)
Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel
ISBN 3–8055–7132–1

............................
Dedication
This book is dedicated to Dr. Lester Packer, Professor of Physiology at
the University of California, Berkeley, since 1961, and his wife Anne.
Together, and each in their own way, they have nurtured the careers of
countless scientists, including four authors of this book.

............................
Contents
IX
Preface
Detection of Free Radicals in Skin
1
Detection of Free Radicals in Skin: A Review of the Literature and
New Developments
Fuchs, J. (Frankfurt); Herrling, T.; Groth, N. (Berlin)
18
Electron Paramagnetic Resonance Detection of Free Radicals in
UV-Irradiated Human and Mouse Skin
Jurkiewicz Lange, B.A. (Neenah, Wisc.); Buettner, G.R. (Iowa City, Iowa)
Detection of Antioxidants in Skin and Antioxidant Response to Environmental Stress
26
The Antioxidant Network of the Stratum corneum
Thiele, J.J.; Schroeter, C.; Hsieh, S.N. (Jena); Podda, M. (Frankfurt);
Packer, L. (Berkeley, Calif.)
43
Activity of Alpha-Lipoic Acid in the Protection against Oxidative
Stress in Skin
Podda, M.; Zollner, T.M.; Grundmann-Kollmann, M. (Frankfurt);
Thiele, J.J. (Jena); Packer, L. (Berkeley, Calif.); Kaufmann R. (Frankfurt)
52
Ozone: An Emerging Oxidative Stressor to Skin
Weber, S.U.; Han, N.; Packer, L. (Berkeley, Calif.)
Oxidative DNA Damage
62
Effects of UV and Visible Radiations on Cellular DNA
Cadet, J.; Douki, T.; Pouget, J.-P.; Ravanat, J.-L.; Sauvaigo, S. (Grenoble)
VII

74
Sequence-Specific DNA Damage Induced by UVA Radiation in the
Presence of Endogenous and Exogenous Photosensitizers
Kawanishi, S.; Hiraku, Y. (Tsu, Mie)
UVA and UVB Induced Signal Transduction in Skin
83
UV-Induced Oxidative Stress and Photoaging
Wenk, J.; Brenneisen, P.; Meewes, C.; Wlaschek, M.; Peters, T.; Blaudschun, R.;
Ma, W.; Kuhr, L.; Schneider, L.; Scharffetter-Kochanek, K. (Cologne)
95
UVA and Singlet Oxygen as Inducers of Cutaneous Signaling Events
Klotz, L.-O.; (Du
¨
sseldorf); Holbrook, N.J. (Baltimore, Md.); Sies, H. (Du
¨
sseldorf)
114
Reactive Oxygen Species as Mediators of UVB-Induced Mitogen-
Activated Protein Kinase Activation in Keratinocytes
Peus, D. (Munich); Pittelkow, M.R. (Rochester, Minn.)
Antioxidant Protection against Oxidative Stress in Skin
128
Antioxidants in Chemoprevention of Skin Cancer
Ahmad, N.; Katiyar, S.K.; Mukhtar, H. (Cleveland, Ohio)
140
Radical Reactions of Carotenoids and Potential Influence on
UV Carcinogenesis
Black, H.S. (Houston, Tex.); Lambert, C.R. (New London, Conn.)
157
Protective Effects of Topical Antioxidants in Humans
Dreher, F.; Maibach, H. (San Francisco, Calif.)
165
The Antioxidative Potential of Melatonin in the Skin
Fischer, T.W.; Elsner, P. (Jena)
175
Bioconversion of Vitamin E Acetate in Human Skin
Nabi, Z.; Tavakkol, A. (Piscataway, N.J.); Dobke, M. (Newark, N.J.);
Polefka, T.G. (Piscataway, N.J.)
187
Author Index
188
Subject Index
VIIIContents

............................
Preface
Are free radicals and reactive oxygen species relevant for dermatopathol-
ogy? Do antioxidants really protect against free-radical-mediated cutaneous
disease and aging? In the past decade, a strongly increasing number of scientific
publications on oxidative stresss and redox regulation in skin indicates the
emerging importance of this field in experimental dermatology.
Furthermore, the increase in scientific evidence for protective antioxidative
mechanisms in the skin has led to a considerably growing interest of the
pharmaceutical and cosmetic industry in therapeutic antioxidant strategies.
Likewise, such terms as ‘free radicals’, ‘antioxidants’ and ‘oxidative stress’
have experienced an almost inflationary use in the lay press and thus raised
enormous interest in the public. Owing to this growing public, academic and
corporate demand, this book is intended to provide an up-to-date overview
of oxidants and antioxidants in cutaneous biology. This book compiles con-
tributions from leading investigators on the detection of free radicals and
antioxidants, their responses to environmental oxidative stressors, the role of
oxidative DNA damage, UVB- and UVA-induced signal transduction, and
antioxidant protection strategies. The chapters mainly focus on the outermost
organ of the body, the skin. Thus, it represents a unique collection of important
new facts and background information on oxidative-stress-related biochem-
istry, photobiology, molecular biology, pharmacology and cosmetology of the
skin. The editors are indebted to all authors for the knowledge and effort they
have invested in this project.
We sincerely hope that this book will provide valuable advice to our
readers and thus will stimulate further the discovery of relevant redox-regulated
pathways and the development of potent therapeutic antioxidant strategies in
dermatology.
Jens Thiele, MD, Jena
Peter Elsner, MD, Jena
IX

Detection of Free Radicals in Skin
Thiele J, Elsner P (eds): Oxidants and Antioxidants in Cutaneous Biology.
Curr Probl Dermatol. Basel, Karger, 2001, vol 29, pp 1–17
............................
Detection of Free Radicals in Skin:
A Review of the Literature and
New Developments
Ju
¨
rgen Fuchs
a
, Thomas Herrling
b
, Norbert Groth
b
a
Department of Dermatology, Medical School, J.W. Goethe University, Frankfurt, and
b
Center of Scientific Instruments, Laboratory for EPR Tomography, Berlin, Germany
It is generally believed that free radicals play an important role in the
pathogenesis of several human diseases. When enough people believe in some-
thing, it becomes known or accepted. However, an increased generation of
free radicals may be the consequence of tissue damage, an epiphenomenon
or only of limited clinical significance. If free radicals are produced in a
clinical condition, it has to be proved that these species are formed and are
an obligatory intermediate for the pathology [1]. Of the several methods avail-
able to study free radicals in biological systems, electron paramagnetic reso-
nance (EPR) spectroscopy is known to be the most important technique.
Using the highly selective EPR technique, free radicals can be detected, charac-
terized and quantified in biological systems. EPR spectroscopy is concerned
with the resonant absorption of microwave radiation by paramagnetic samples
in the presence of an applied magnetic field. Free radicals are paramagnetic
species due to the unpaired electron in the outer orbit and have a magnetic
moment. If an external magnetic field is applied to these molecules, their axes
are directed either parallel (energetically more stable) to the external field or
in the opposite direction (antiparallel). If electromagnetic waves which match
the energy difference between the parallel and antiparallel electronic moments
(microwaves) are applied to this system, a change in the orientation of these
molecules will occur. The net absorption of the microwave energy under these
resonance conditions is quantitated and the 2nd derivative is recorded as the
EPR signal. Soon after the discovery of the EPR methodology by the Russian
student Zavoiksy in 1945 [2], the method was applied for detection of free
radicals in biological samples [3]. Since then, an uncountable number of reports

on the development and application of EPR spectroscopy in biomedicine has
appeared. One of the most comprehensive compilations of the EPR literature
was given annually in the ‘Specialist Periodical Reports by the Royal Society
of Chemistry’ [4]. The skin is a target organ of oxidative stress; because it is
continuously exposed to high oxygen concentrations and solar radiation, it
serves as a major portal of entry for many oxidizing environmental pollutants
and occupational hazards, and contains several readily oxidizable molecules
critical for structure and function. For these and other practical reasons out-
lined below, free radical biologists are becoming more and more interested in
cutaneous EPR applications. The purpose of this review is to provide the
reader with current information on the developments of free radical detection
in skin by the EPR methodology and other applications of the EPR technique
in cutaneous biology.
Direct Detection of Free Radicals
Directevidence for free radicalformationinhumanandanimal skin follow-
ing exposure to UV radiation has been obtained by low-temperature (e.g.
–196 ºC) EPR spectroscopy in vitro [5–7]. At the temperatureof liquid nitrogen,
the radical steady-state concentration is high enough to give a sufficient EPR
signal intensity. However, these signals are very broad and usually provide no
or only very limited information about the chemical identity of the free radical
structure. At ambient temperature, only persistent free radicals such as melanin
[8–14]or the ascorbylradical [15–18]havebeen directly detectedin skin and/orin
skin appendages in vitro. The cutaneous metabolism of free-radical-generating
compounds such as 4-hydroxyanisole and anthralin has been shown to generate
persistent semiquinone radicals in rat skin [19] or anthrone/anthrone-dimer-
derived radicals in pig or mouse skin in vitro [20, 21], respectively. Tocopheroxyl
radicals were detected in vitamin-E-treated mouse skin in vitro after UV irradi-
ation[22]. Someinvestigators haveanalyzedlyophilizedor abradedskinsamples
(e.g. horn) for free radicals. The freeze-dry technique and mechanical processes
such as cutting and grinding can produce paramagnetic artifacts in biological
tissues [23, 24]. These techniques should be avoided, because they can lead to
erroneous results in EPR experiments.
Indirect Detection of Free Radicals
In most cases, the low steady-state concentration of reactive free radicals
in biological samples allows only indirect detection by the spin trapping
2Fuchs/Herrling/Groth

method. Spin trapping is defined as that chemical reaction in which a radical
adds to a molecule so that the group that was the radical (radical addend)
stays with the molecule for future analysis. The molecule which captures the
radical is called the spin trap. The additional product is called the spin adduct.
Spin traps are usually nitrones or nitroso compounds, which are one electronic
oxidation state above the nitroxides and which, upon reacting with free radicals,
become converted to nitroxides. The trapped radical has a characteristic EPR
spectrum that allows chemical identification of the highly reactive free radical.
The interpretation of the EPR spectra and the subsequent identification of
the free radical adduct is the most sophisticated and difficult component of
the spin trapping process [1, 25, 26]. The stability of the radical adduct and
its conversion into other paramagnetic or diamagnetic products can be a
source of experimental confusion. The history of spin trapping began with
C-phenyl-N-tert-butylnitrone (PBN), which is used for detection of carbon-
and oxygen-centered free radicals [1, 26]. The capabilities of PBN were im-
proved by synthesizing the pyridine-N-oxide analogue of PBN, C-(4-pyridinyl-
N-oxide)-N-tert-butylnitrone (4-POBN). Dithiocarbamate iron (II) complexes
are successfully used for spin trapping NO in biological systems [27, 28],
while 2,2,6,6-tetramethyl-piperidine and 2,2,6,6-tetramethyl-4-piperidone are
sensitive trapping agents for singlet oxygen [29, 30]. 5,5-Dimethyl-1-pyrroline-
N-oxide (DMPO) is probably the most widely used spin trap in biological
systems, scavenging carbon-, oxygen- and sulfur-centered free radicals [1, 27].
Table 1 shows some selected examples of spin trapping in keratinocytes, epi-
dermis homogenate and skin biopsies. The free radical xenobiotic metabolism
can be investigated by the spin trapping technique in animals in vivo, and
some selected examples of in vivo spin trapping are shown in table 2. To
our knowledge however, until now no in vivo studies in skin have yet been
published.
Nitroxide-Based Electron Paramagnetic Resonance
Because of their physicochemical versatility, nitroxides can be used as
imaging agents for a number of different purposes including the investigation
of the cellular redox status, structural and dynamic properties of biological
membranes, oximetry and pH measurements.
Redox Measurements
Nitroxides can be used for the study of redox metabolism [45–47]. Nitrox-
ide free radicals accept electrons from a variety of sources such as low-molecu-
lar-weight antioxidants, certain enzymes such as the cytochrome P
450
system,
3Electron Paramagnetic Resonance Applications on Skin

Table 1. In vitro spin trapping in keratinocytes and epidermis homogenate
Free radical or reactive Sample Spin trap Reference
species detected
Alkyl hydroxyl rat epidermis homogenate DMPO 7
Lipid alkyl mouse skin biospy DMPO 17
Lipid alkoxyl human skin biopsy 4-POBN
Alkyl mouse skin biopsy DMPO 31
Alkoxyl
Singlet oxygen human bronchial epithelial cells 2-(9,10-dimethoxyanthracenyl)- 32
tert-butylhydroxylamine
Glutathione thiyl keratinocytes DMPO 33
Alkyl mouse keratinocytes DMPO 34
Alkoxyl
Hydroxyl murine skin fibroblasts DMPO 35
Methyl human keratinocytes 4-POBN 36
Methyl human squamous carcinoma 3,5-dibromonitrosobenzene 37
keratinocytes sulfonic acid
Hydroxyl guinea pig epidermis homogenate DMPO 38
Lipid alkyl hydroxyl rat epidermis homogenate DMPO 39
Carbon-centered mouse skin biopsy 4-POBN 40
the mitochondrial respiratory chain and transition metal ions. They also react
with reactive oxidants, such as the superoxide anion radical [48, 49], and other
free radical species [50]. Thus, the biokinetics of nitroxides is sensitive to the
reducing as well as to the oxidizing activity of their ultimate surrounding. The
distribution of the nitroxides as well as the heterogenous cellular and subcellu-
lar distribution of different reducing and oxidizing agents must be taken into
account, when analyzing nitroxide biokinetics. Nitroxide-based in vitro EPR
studies for the measurement of redox components in isolated keratinocytes,
skin homogenates and intact skin samples have been published [51–55]. As
outlined above, the signal decay rate of nitroxides is enhanced by oxidative
stress, and this enhanced decay can be suppressed by administration of antioxi-
dants. Due to their redox properties 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolid-
ine-N-yloxyl (CTPO) is preferentially used to study the effect of reactive
oxidants, while 2,2,6,6-tetramethyl-piperidine-N-oxyl (TEMPO) is employed
4Fuchs/Herrling/Groth

Table 2. In vivo spin trapping
Reactive species Sample Spin trap EPR Reference
technique
Sulfur trioxide whole-mouse sequential DMPO 1 GHz 41
anion radical intravenous injections of
sodium sulfite and sodium
dichromate
Oxygen-centered blood effluate from ischemic DMPO 1 GHz 42
radicals skin flap
Nitric oxide subcutaneous compartment N-(dithiocarboxy) 1 GHz 43
of mice, injected with sarcosine
isosorbite dinitrate
Hydroxyl radical subcutaneous mouse tumor, DMPO 1 GHz 44
ionizing irradiation
for measurement of tissue antioxidant activity. Nitroxides have been used as
indicators of the cellular redox status for in vivo measurement of antioxidant
status as well as oxidative stress as shown in table 3.
Oximetry
EPR studies can give information on the tissue oxygen tension by virtue
of a physical interaction of molecular oxygen, which is paramagnetic, and a
spin probe thereby modifying the spectral characteristic of the spin probe.
The extent of spectral broadening can usually be directly correlated to oxygen
concentration by appropriate calibrations. Spin probes that have been used for
tissue oximetry include nitroxides, lithium phthalocyanine, India ink, fusinite,
synthetic chars and coals. This method provides a sensitivity, accuracy and
range to measure physiologically and pathologically pertinent oxygen tensions
in vivo. For illustration, a solid-state paramagnetic probe such as lithium
phthalocyanine [66] or charcoal [67] has been used in localized tissue oximetry,
and nitroxide spin probes were used for measurement of tissue oxygen tension
in ischemic tissue [68]. Low-frequency EPR (250 MHz) was successfully utilized
to measure oxygen tension in tumor tissues of living mice using a perdeuterated
nitroxide spin probe [69]. To date there are only a few reports of EPR oximetry
in skin [70, 71]. It seems likely that this will change in the near future, because
skin oxygenation greatly influences the effectiveness of many anticancer thera-
pies such as chemotherapy, radiation therapy, hyperthermia and photodynamic
therapy. The effectiveness of these therapies can be manipulated by modulating
5Electron Paramagnetic Resonance Applications on Skin

Table 3. Nitroxides as indicators of the cellular redox status for in vivo measurement
of antioxidant status as well as oxidative stress
Antioxidant/oxidant modulation Nitroxide Sample EPR technique Reference
of tissue redox status
Antioxidant supplementation TEMPO mouse lung 1 GHz 56
Antioxidant supplementation TEMPO rat 1 GHz 57
Antioxidant status UV irradiation TEMPO human skin 3 GHz 58
Streptozotocin-induced diabetes CTPO rat 300 MHz 59
Silica-induced lung injury TEMPO rat lung 1 GHz 60
Hyperoxia CTPO mouse 1 GHz, loop gap 61
resonator
Ionizing radiation CTPO mouse 1 GHz, loop gap 62
resonator
Iron overload CTPO mouse 1 GHz, loop gap 63
resonator
Ionizing radiation CTPO mouse 1 GHz 64
Carbon tetrachloride CTPO mouse 1 GHz 65
skin oxygen tension. It may be possible to utilize EPR-based oximetry as an
in situ predictor of the energy/dose required to elicit a biological response in
skin.
Membrane Structure
Spin labeling is an EPR technique used to monitor biophysical properties
of biological membranes. For example, this is achieved by introducing a nitrox-
ide-labeled fatty acid into the membrane system. The nitroxide group is sensi-
tive to its biophysical surrounding. Thereby fluidity and polarity of membranes
can be analyzed in complex biological samples. Since the lamellarly arranged
lipid bilayers of the stratum corneum control the diffusion and penetration
of chemical substances into and through the skin, EPR-based measurement
of stratum corneum lipid microviscosity and polarity provides information on
the barrier function of the epidermis. An increase in fluidity of skin lipid
bilayers suggests a decrease in the skin barrier function. The pH value of the
stratum corneum is an important regulating factor for the stratum corneum
homeostasis, and it is assumed that the pH is among the factors that regulate
the integrity of the skin barrier function. The use of pH-sensitive nitroxides,
6Fuchs/Herrling/Groth

in conjunction with EPR, offers a unique opportunity for noninvasive assess-
ment of pH values in vivo |72]; however, no such studies have yet been per-
formed in skin. The fluidity of animal and human skin-derived stratum
corneum lipids was analyzed in vitro by the EPR technique employing nitrox-
ide-labeled 5(12,16)-doxylstearic acid [3–77] and perdeuterated di-tert-butyl-
nitroxide [78]. It was suggested that EPR may provide a facile and robust
method to define the subclinical irritancy potential of chemicals [75].
Electron Paramagnetic Resonance Imaging
The spatial distribution of free radicals within a biological sample can
be analyzed by utilizing magnetic field gradients in a manner similar to that
of NMR imaging [79, 80]. EPR imaging (EPRI) and NMR imaging are based
on similar principles. However, the superiority of NMR imaging is due to its
excellent sensitivity. In vivo EPRI is presently restricted to artificial free radicals
introduced into a biological sample with a sensitivity of tissue free radical
concentration less than 0.1 mM compared to more than 100 M of endogenous
proton concentrations available for NMR. EPRI can be performed in the
spatial range to obtain one-, two- or three-dimensional images of free radical
distribution in samples. The imaging technique that also includes a spectral
dimension is termed spectral-spatial imaging. Spectral-spatial imaging can
also be performed in one, two or three spatial dimensions. The spectral-spatial
image contains more information, but it is technically more difficult to obtain.
We have used the EPRI technique with modulated field gradients to obtain
spatial resolution of paramagnetic centers in different tissue planes. This
approach allows the measurement of an EPR spectrum in a selected volume
part [81]. EPRI at 9 GHz was used in vitro for measuring biokinetics and spatial
or spectral-spatial distribution of nitroxides in mouse or pig skin [82–87].
Penetration of nitroxide-labeled drugs such as retinoic acid and dihydrolipoate
was studied by this technique in mouse skin biopsies [88, 89]. Although there
are some limitations of the spin labeling method, drug penetration as well as
drug-membrane and drug-enzyme interactions in skin can be investigated by
labeling pharmacologically active compounds.
The working frequency, the radical concentration and the magnetic field
gradient determine the spatial resolution of EPRI. The application of a single
line paramagnetic label yielded an image resolution better than 100 lmat
1 GHz on samples of up to 20 mm in size [90]. Berliner et al. [91] mea-
sured for the first time in vivo an EPR image of a murine tumor (Cloudman
S-91 melanoma in the tail of a DBA-2J mouse) using nitroxide injected into
the tail vein. They obtained a cross-sectional image perpendicular to the tail
7Electron Paramagnetic Resonance Applications on Skin

Fig. 1. Penetration depth of microwaves into biological tissues as a function of different
frequencies.
axis, which clearly distinguished features to the submillimeter resolution level
[91]. The application of the EPRI technique in vivo to obtain high-quality
images of biological samples is presently limited by several factors including
gradient design and accuracy, sensitivity as well as speed of acquisition. A
further intrinsic problem of the EPRI technique is the short relaxation time
of the free electron. The line width associated with EPR signals is 3 orders
of magnitude larger compared to that of NMR signals. Therefore EPRI
requires 100–1,000 times more powerful gradients, and this significantly
decreases the sensitivity, requiring a tissue spin probe concentration of at
least 0.1 mM.
In vivo Electron Paramagnetic Resonance
In vivo applications of EPR are hampered by the limited tissue penetration
depths of microwaves and the high nonresonant dielectric loss of the exciting
frequency. Due to the high water content of biological samples, the penetration
depth of the most commonly applied microwave frequency of 9 GHz is less
than 1 mm [92]. Figure 1 shows the penetration depth of microwaves into
biological tissues as a function of different frequencies, indicating that low
microwave frequencies have a better tissue penetration than high-frequency
radiation. The nonresonant dielectric absorption of microwaves in biological
samples is a function of the frequency. 9-GHz EPR measurements on lossy
samples are limited to a volume of few microliters, 1- to 3-GHz measurements
8Fuchs/Herrling/Groth

are possible on volumes of a few milliliters and radiofrequency resonators
(200–300 MHz) are used for samples of up to 200 ml. Depending on the
special characteristics of the EPR instrument and sample cavity used, skin
biopsies 4–6 mm in diameter are the maximum size that allows for adequate
tuning using a conventional H
1
0
2
cavity at 9 GHz frequency [93]. The strength
of the EPR signal is proportional to the product of the quality factor and the
filling factor of the cavity. Resonator cavity design is critical to achieve max-
imum sensitivity and must be adapted to accommodate the sample with the
highest possible filling factor¶quality factor product. The application of loop
gap and reentrant resonators provides an excellent approach to achieve this.
Surface coil type loop gap resonators were successfully used for imaging the
subcutaneous compartment of mice [94] or subcutaneously localized tumors
in mice [95] in vivo. Due to their better tissue-penetrating properties, microwave
frequencies lower than 3 GHz permit in vivo EPR studies in whole animals
[96, 97]. Whole-body EPRI in small animals measuring nitroxide radical
elimination was successfully performed at very low microwave frequency (200–
700 MHz) [98, 99] and at low frequency (1 GHz) [100, 101]. However, a severe
disadvantage of reducing the microwave frequency is a significant loss in
sensitivity. The sensitivity of the measurement is directly related to the square
of the operating frequency. For practical reasons, at 9 GHz frequency, the
detectability limit of EPR spectroscopy using a conventional H
1
0
2
cavity is in
the range of 10
–6
–10
–7
M, although lower radical steady-state concentrations
can be detected in biological samples by accumulation techniques, modifica-
tions of the cavity or more sensitive detection methods. A further loss in
sensitivity (100- to 1,000-fold) occurs by application of the imaging technique
in comparison to spectroscopy, depending on the image reconstruction
method. This explains that the usually applied nitroxide concentrations in
small animal in vivo EPRI experiments are in the range of 0.1–10 mM and
higher. Technical innovations of in vivo EPR have been the integration and
combination of different magnetic resonance techniques, such as the develop-
ment of proton electron double resonance imaging (PEDRI) and field-cycled
dynamic nuclear polarization (FC-DNP). At low microwave frequency PEDRI
and FC-DNP analyze the electron spins indirectly via their effect on a proton
NMR signal [102–104]. Both techniques require administration of artificial
spins (e.g. nitroxide radicals) and almost all the machines used for PEDRI
and FC-DNP have been constructed for small animals. As already outlined
above, the most restricting factors for clinical applications are the limited
tissue penetration of the microwaves and the weak EPR effect of low-frequency
microwave radiation causing a significant loss in sensitivity. An approach to
improve the sensitivity is the development of new detection techniques for
the EPR resonance phenomenon such as the design of instrumentation with
9Electron Paramagnetic Resonance Applications on Skin

Table 4. In vivo skin EPR spectroscopy and imaging
Nitroxide Organ Species EPR technique Reference
TEMPO skin human 3-GHz spectroscopy 58
CTPO melanoma mouse 1.5-GHz imaging 83
Polynitroxyl albumin+ subcutaneous mouse 1-GHz spectroscopy and imaging 94
4-hydroxy-2,2,6,6-tetramethyl- tissue
piperidine-N-oxyl
Perdeuterated N
15
TEMPO and subcutaneous mouse 1-GHz spectroscopy and imaging 95
CTPO tissue
Perdeuterated N
15
CTPO skin mouse 3-GHz spectroscopy 107
TEMPO skin human 3-GHz spectroscopy 108, 109
CTPO skin mouse 9-GHz spectroscopy 110
Di-tert-butylnitroxide tail vasculature mouse 9-GHz spectroscopy 111
CTPO subcutaneous rat 700-MHz spectroscopy, flexible 112
tissue surface coil type resonator
(stethoscope-like application)
Anthralin-derived free radicals skin mouse 1-GHz spectroscopy 113
Cr(V) skin rat 1-GHz spectroscopy 114
longitudinally detected EPR [105]. Other techniques comprise photoacoustic
detection of magnetic resonance, detecting magnetic flux changes due to reso-
nance by a superconductive split ring, Raman-heterodyne EPR, electrical
detection of EPR signals (ED-EPR and STM-EPR) and fluorescence-detected
magnetic resonance [106]. ED-EPR, STM-EPR and fluorescence-detected
magnetic resonance have been used for the detection of very strong paramag-
netic centers such as in ferritin or silicium, but it is questionable whether these
techniques can be applied to biological samples. However, further develop-
ments of these detection techniques may lead to a significant improvement in
the sensitivity, which is presently a limiting factor for many EPR applications
in vivo.
Because of its location, the skin is fully accessible to relatively higher-
frequency EPR (eg. 3–9 GHz), in contrast to many other sites where the
depth of sensitivity can be limiting. Presently, the skin is the only human
organ which can be measured in vivo with sufficient sensitivity. Table 4 shows
some selected examples of in vivo EPR spectroscopy and imaging in animal
10Fuchs/Herrling/Groth

and human skin. Two different experimental arrangements have been used
for in vivo measurements on skin, and one technique is presently being
developed for localized skin EPR measurements of the human body. For the
microwave system, either a 9-GHz bridge with a cavity or a 3-GHz bridge
with a surface coil are used. The 9-GHz bridge system is based on the concept
of Furusawa and Ikeya [115], who used microwave cavities with a small hole
from which the microwave field leaks out to a small cross-sectional area of
the object. This method is also applicable for localized spectroscopy on skin.
For the magnet system, a normal electromagnet with a gap of 100 mm is
wide enough to accommodate human limbs between the pole faces. Figure 1
shows that the microwave penetration depths lie between 0.5–1.0 mm (9 GHz)
and about 5 mm (3 GHz), respectively. This means that 9 GHz is restricted
to the upper layer of the skin (i.e. the human epidermis and upper dermis,
or total mouse skin). For deeper layers (i.e. the full human dermis or subcutis)
penetration depths of 5 mm (3 GHz) or 3.5 cm (1 GHz) are more suited,
respectively. The probe head is a 90-degree bent surface coil (8 mm diameter)
with an electronically matched system. A quartz plate is mounted on one
side of the surface coil, which defines a plane-parallel measuring area on
the skin. Matching is accomplished by placing a piezoelectric element at a
distance of
l
/
4
outside the loop. Two 100-kHz modulation coils near the
surface coil generate a modulation field Bm in the skin layer. The rapid scan
coils are mounted on the surface of the magnet pole plates. For human
measurements, the probe head is mounted on the forearm placed in the
magnet. It is important to point out that this apparatus is restricted to human
limbs and does not allow measurements on other parts of the body such as
the breast or spine which would require a more flexible probe head accessible
to all parts of the body. Since magnetic field requirements for EPR, such as
strength, homogeneity and stability of the magnetic field, are much lower
than for NMR, this allows the use of a compact and flexible probe head of
the size used in sonography, easily permitting skin measurements on all parts
of the human body without requiring a whole-body magnet. Such a flexible
compact probe head is presently under construction in our laboratory. The
prototype of this newly designed probe head consists of two rectangular
pieces of Neo Delta Magnet material (Nd Fe B), connected by an iron
backbone. Two small magnet pieces near the microwave loop increase the
homogeneity of the magnetic field in the upper skin layers. A field sweep of
6 mT is generated by two coils wound on the backbone of the magnet system.
The microwave shield above the microwave loop prevents interactions between
the loop and the magnet. This flexible and compact probe head will allow
nitroxide and spin-trap-based EPR spectroscopy in vivo on all parts of the
human skin with high specificity.
11Electron Paramagnetic Resonance Applications on Skin

Outlook
The growing interest in the role of free radicals in the pathogenesis of
human diseases has led to an increased need for techniques to measure free
radicals in the clinical situation [116]. EPR spectroscopy is a highly selective
assay for detecting free radicals and is the most important technique for
characterization and quantification of these species in biological systems. EPRI
can be performed in the spatial range to obtain images of free radical distribu-
tion in samples, while spectral-spatial imaging includes a spectral dimension.
The success of EPR-based studies relies heavily on the use of spin traps or
nitroxides, because the steady-state concentration of most endogenous free
radicals is orders of magnitude below the detectability limit of EPR. Although
in the last few years EPR spectroscopy and EPRI techniques have been con-
siderably developed to give useful biochemical and biophysical information
in vivo, these methodologies need to be improved for reliable application under
clinical conditions, as several intrinsic technical problems must still be resolved.
Human in vivo EPR is hampered by a significant loss in sensitivity when using
more deeply penetrating low-frequency microwaves and by practical problems
of the sample and magnet/resonator size. It was guessed that at least one or
two orders of magnitude of higher sensitivity are necessary to detect reactive
free radicals directly in tissue [106]. However, this is presumably an optimistic
calculation. Improved detection techniques and the development of high-
quality surface coil resonators could help to resolve some of the difficulties.
The use of in vivo EPR to study metabolic processes in skin appears to be
an attractive and effective approach, because of the importance of this organ
and its acccessibility. The skin is presently the only human organ which can
be measured in vivo with sufficient sensitivity at 9 and 3 GHz frequency. The
more widespread application of localized EPR spectroscopy in human skin
in vivo will significantly contribute to improve our understanding of cutaneous
free radical processes and redox biochemistry. Furthermore, the EPR methodo-
logy is a useful tool for the noninvasive in vivo measurement of skin barrier
function, drug/skin interaction and cutaneous oxygen tension.
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