Skin, Hair, and Nails, Forslind, 2005

Skin, Hair, and Nails
BASIC AND CLINICAL DERMATOLOGY

Series Editors
ALAN R.SHALITA, M.D.
Distinguished Teaching Professor and Chairman
Department of Dermatology
State University of New York
Health Science Center Brooklyn
Brooklyn, New York
DAVID A.NORRIS, M.D.
Director of Research
Professor of Dermatology
The University of Colorado
Health Sciences Center
Denver, Colorado
1. Cutaneous Investigation in Health and Disease: Noninvasive Methods and
Instrumentation, edited by Jean-Luc Lévêque
2. Irritant Contact Dermatitis, edited by Edward M.Jackson and Ronald Goldner
3. Fundamentals of Dermatology: A Study Guide, Franklin S.Glickman and Alan
R.Shalita
4. Aging Skin: Properties and Functional Changes, edited by Jean-Luc Lévêque
and Pierre G.Agache
5. Retinoids: Progress in Research and Clinical Applications, edited by Maria
A.Livrea and Lester Packer
6. Clinical Photomedicine, edited by Henry W.Lim and Nicholas A.Soter
7. Cutaneous Antifungal Agents: Selected Compounds in Clinical Practice and
Development, edited by John W.Rippon and Robert A.Fromtling
8. Oxidatlve Stress in Dermatology, edited by Jürgen Fuchs and Lester Packer
9. Connective Tissue Diseases of the Skin, edited by Charles M.Lapière and
Thomas Krieg
10. Epidermal Growth Factors and Cytokines, edited by Thomas A.Luger and
Thomas Schwarz
11. Skin Changes and Diseases in Pregnancy, edited by Marwali Harahap and
Robert C.Wallach
12. Fungal Disease: Biology, Immunology, and Diagnosis, edited by Paul H.
Jacobs and Lexie Nall
iii
13. Immunomodulatory and Cytotoxic Agents in Dermatology, edited by Charles

J.McDonald
14. Cutaneous Infection and Therapy, edited by Raza Aly, Karl R.Beutner, and
Howard I.Maibach
15. Tissue Augmentation in Clinical Practice: Procedures and Techniques, edited
by Amold William Klein
16. Psoriasis: Third Edition, Revlsed and Expanded, edited by Henry H.
Roenigk, Jr., and Howard I.Maibach
17. Surgical Techniques for Cutaneous Scar Revision, edited by Marwali
Harahap
18. Drug Therapy in Dermatology, edited by Larry E.Millikan
19. Scarless Wound Healing, edited by Hari G.Garg and Michael T.Longaker
20. Cosmetic Surgery: An Interdisciplinary Approach, edited by Rhoda S. Narins
21. Topical Absorption of Dermatological Products, edited by Robert L.
Bronaugh and Howard I.Maibach
22. Glycolic Acid Peels, edited by Ronald Moy, Debra Luftman, and Lenore
S.Kakita
23. Innovative Techniques in Skin Surgery, edited by Marwali Harahap
24. Safe Liposuction, edited by Rhoda S.Narins
25. Psychocutaneous Medicine, edited by John Y.M.Koo and Chai Sue Lee
26. Skin, Hair, and Nails: Structure and Function, edited by Bo Forslind and
Magnus Lindberg
ADDITIONAL VOLUMES IN PREPARATION
Vitiligo: Problems and Solutions, edited by Torello Lotti and Jana Hercogova
Itch: Basic Mechanisms and Therapy, edited by Gil Yosipovitch, Malcolm
W.Greaves, Alan B.Fleischer, and Francis McGlone
Structure and Function
edited by
Bo Forslind
Karolinska Institutet
Stockholm, Sweden
Magnus Lindberg
Stockholm Center of Public Health
and Karolinska Institutet
Stockholm, Sweden
Associate Editor
Lars Norlén
University of Geneva
Geneva, Switzerland
MARCEL DEKKER, INC. NEW YORK • BASEL

This edition published in the Taylor & Francis e-Library, 2005.
“To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of
thousands of eBooks please go to www.eBookstore.tandf.co.uk.”
Although great care has been taken to provide accurate and current information,
neither the author(s) nor the publisher, nor anyone else associated with this
publication, shall be liable for any loss, damage, or liability directly or indirectly
caused or alleged to be caused by this book. The material contained herein is not
intended to provide specific advice or recommendations for any specific situation.
Trademark notice: Product or corporate names may be trademarks or registered
trademarks and are used only for identification and explanation without intent
to infringe.
Library of Congress Cataloging-in-Publication Data
A catalog record for this book is available from the Library of Congress.
ISBN 0-203-91286-1 Master e-book ISBN
ISBN: 0-8247-4313-X (Print Edition)
Headquarters
Marcel Dekker, Inc., 270 Madison Avenue, New York, NY 10016, U.S.A.
tel: 212–696–9000; fax: 212–685–4540
Distribution and Customer Service
Marcel Dekker, Inc., Cimarron Road, Monticello, New York 12701, U.S.A.
tel: 800–228–1160; fax: 845–796–1772
Eastern Hemisphere Distribution
Marcel Dekker AG, Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland
tel: 41–61–260–6300; fax: 41–61–260–6333
World Wide Web
http://www.dekker.com
The publisher offers discounts on this book when ordered in bulk quantities. For
more information, write to Special Sales/Professional Marketing at the headquarters
address above.
Copyright © 2004 by Marcel Dekker, Inc. All Rights Reserved.
Neither this book nor any part may be reproduced or transmitted in any form or by
any means, electronic or mechanical, including photocopying, microfilming, and
recording, or by any information storage and retrieval system, without permission in
writing from the publisher.
Series Introduction
Over the past decade, there has been a vast explosion in new information relating
to the art and science of dermatology as well as fundamental cutaneous biology.
Furthermore, this information is no longer of interest only to the small but
growing specialty of dermatology. Scientists from a wide variety of disciplines
have come to recognize both the importance of skin in fundamental biological
processes and the broad implications of understanding the pathogenesis of skin
disease. As a result, there is now a multidisciplinary and worldwide interest in
the progress of dermatology.
With these factors in mind, we have undertaken to develop this series of books
specifically oriented to dermatology. The scope of the series is purposely broad,
with books ranging from pure basic science to practical, applied clinical
dermatology. Thus, while there is something for everyone, all volumes in the
series will ultimately prove to be valuable additions to the dermatologist’s
library.
The latest addition to the series, by Bo Forslind, Magnus Lindberg, and Lars
Norlén, is both timely and pertinent. We trust that this volume will be of broad
interest to scientists and clinicians alike.
Alan R.Shalita
SUNY Health Science Center
Brooklyn, New York
In Memoriam: Bo Forslind
This book is a result of Bo Forslind’s initiative and work. Bo started his medical
studies in the late 1950s, undertaking research projects and education at the
Department of Medical Biophysics at Karolinska Institutet in Stockholm,
Sweden. In biophysics it was possible for Bo to combine a genuine interest in
physics and chemistry with a desire to solve problems in medicine. Early in his
career, he was recognized internationally for his biophysical studies on hair and
nails. After this he expanded his interests to include the skin itself. Throughout
his career, Bo applied new methods— such as electron microscopy,
autoradiography, and X-ray diffraction—to analyze and solve medical problems.
Over the past 25 years, Bo was a pioneer in applying electron- and proton-
induced X-ray microanalysis (EDX and PIXE) in studies on skin, hair, and nails.
During the past decade most of Bo’s research energy was focused on the stratum
corneum and the structure and function of the skin barrier. In 1994, for example,
he presented an important model, the domain mosaic model, for describing the
arrangement of the stratum corneum and its lipids.
Bo was an open-minded researcher who established contacts with other
researchers throughout the world. He was also an appreciated lecturer and
mentor for research students. Bo was active in his department at Karolinska
Institutet throughout his career. Even after his retirement a couple of years ago,
he continued his research. At this time he saw the opportunity at last to edit a
book on hair, nails, and skin. Bo wanted the book to present a structure-function
perspective with a biophysical touch, including basic information as well as new
research results. He also wanted to invite younger scientists to contribute. Using
his contacts (and friendships) with researchers throughout the world, he was able
to gather a highly qualified group. Sadly, Bo would not see the result of this
work. He suddenly became very ill and died of cancer within a couple of
months. However, he was very determined that the book be published. Now,
with the book in print, we see it as a tribute to Bo and his work.
Magnus Lindberg
Lars Norlén
Foreword
The integument—our first line of defense against a hostile physical environment

—was regarded in the past as little more than a passive barrier against
dehydration, physical insults, and microbial invasion. In recent decades,
however, intensive research has redefined the integument as a biologically active
tissue that communicates intimately with the body interior (via immune and
hormonal systems) and regulates its homeostasis (via autoregulatory mechanisms
that sense the environment). This new body of knowledge has emerged as the
product of diverse research efforts using biological, chemical, physical, and
molecular genetic techniques. Further, the combined efforts of biologists,
physicists, and medical doctors in describing the delicate interactions between
different components of the skin and its adnexal structures (i.e., hair and nails)
has enabled application of this knowledge in clinical situations—especially when
the skin barrier fails, as in irritative skin reaction and genetic or immune-
mediated diseases characterized by abnormal skin, hair, or nails. The benefits of
such close interaction between the basic sciences and clinical research are well
illustrated herein by the editors and contributors of this book.
Professor Bo Forslind was, for 40 years, at the forefront of research on the
skin barrier and the structure of hair and nails. His groundbreaking work was
made possible by his insight as a physician and an expert on electron microscopy.
For many years, Professor Magnus Lindberg has been working to bridge the gap
between basic research in skin biology and clinical observations in occupational
dermatology. He is also an expert in studying the skin barrier using noninvasive
techniques. Lars Norlén is the youngest member of the research group founded
by Professor Bo Forslind more than 20 years ago. His work focuses on
epidermal lipids and their composition, as well as the structure and function of
the stratum corneum.
By inviting an international community of experts to contribute to this book,
the editors have consolidated emerging techniques (both applied and
experimental) and theoretical concepts that represent the vanguard of
dermatology. Today, the demand for knowledge about the integument is greater
and the potential for treating diseases and improving health care is clearer than
ever before; to that end, this book presents a thorough, multidisciplinary survey
of new and existing areas of cutaneous research. Because of its broad scope, the
ix
book should serve as an up-to-date reference for scientists in several disciplines

that share a common interest in understanding skin biology and improving the
health of patients affected by skin disease.
Anders Vahlquist, M.D., Ph.D.
Professor of Dermatology
University of Uppsala
Uppsala, Sweden
Preface
The skin and its appendages (i.e., hair and nails) constitute a fantastic organ. Skin
has always been an important arena for interpersonal communication, and it has
been manipulated in various ways, as in tattoos and cosmetics, since the earliest
days of the human race. Nearly as old as this desire to adorn the skin is the desire
to understand its aberrations; descriptions of skin diseases can be found in many
ancient texts.
In both clinical and investigative work, skin is easy to observe; it is also quite
convenient to obtain samples of the skin and its appendages for further analysis.
Skin cells can also be studied in cell and organ cultures. In biological and
medical sciences, skin is an experimental model that is appropriate and easy to
use. The skin can express most forms of pathological changes, ranging from
inflammation, allergy, and tumors to genetic disturbances. During recent decades
it has also become an important route for drug delivery (i.e., transdermal
delivery).
With the ongoing development of innovative investigative techniques, our
knowledge about the integument has increased rapidly. However, a better
understanding of the normal physiology of the skin and the alterations induced
by various pathological mechanisms requires a deeper knowledge about the
relationship between structure and function. In terms of structure and function,
there are many similarities between skin, hair, and nails. However, there are few
texts in the literature that integrate these aspects. This book fills this gap by
offering several chapters dealing with structure and function and others dealing
with the functional changes induced by environmental factors. The contributors
include experts in the front line of research on skin, hair, and nails, as well as
noted comparative biologists and skin pathologists. We believe that this book
should be of interest and value to anyone engaged in skin research in its broadest
sense.
Magnus Lindberg
Lars Norlén
Contents
Series Introduction vi
Foreword viii
Anders Vahlquist
Preface x
Contributors xiii
1. An Introduction to the Book 1

Bo ForslindMagnus Lindberg
Part I Skin
2. Structure and Function of the Skin Barrier: An Introduction 9
Bo ForslindMagnus Lindberg
3. Lipid Phase Behavior: A Basis for an Understanding of 21
Membrane Structure and Function
Emma SparrSven Engström
4. Stratum Corneum Lipid Structure: Insights from NMR and 57
FTIR Spectroscopic Studies
Ya-Wei HsuehJenifer ThewaltNeil KitsonDavid J.Moore
5. “Confidence Intervals” for the “True” Lipid Composition of 75
the Human Skin Barrier?
Philip WertzLars Norlén
6. Stratum Corneum Lipid Organization In Vitro and In Vivo 97
as Assessed by Diffraction Methods
Gonneke S.K.PilgramJoke A.Bouwstra
7. The Mammalian Skin Barrier: Structure, Function, and 139
Formation Considerations
Lars Norlén
8. The Skin Barrier: An Evolutionary and Environmental 173
Perspective
Gopinathan K.Menon
xii
9. Skin Barrier Function in Diseased Skin and in Normal Skin 199

Exposed to Delipidizing Compounds: A Skin Penetration
Perspective
Anders BomanMagnus Lindberg
10. Understanding the Irritative Reaction 215
Carolyn WillisMagnus Lindberg
Part II Hair
11. Formation and Structure: An Introduction to Hair 251
Magnus LindbergBo Forslind
12. The Hair Fiber Surface 263
Leslie N.Jones
13. Biology of Hair Pigmentation 295
Desmond J.Tobin
14. Androgen Influence on Hair Growth 337
Valerie Anne Randall
15. Alopecia Areata: An Update on Etiology and Pathogenesis 359
Andrew J.G.McDonaghRachid Tazi-AhniniAndrew
G.Messenger
Part III Nails

16. The Structure and Properties of Nails and Periungual 377
Tissues
David de BerkerBo Forslind
Index 435
About the Editors 433
Contributors
Anders Boman, Ph.D. Occupational and Environmental Medicine, Stockholm

Center of Public Health, and Occupational and Environmental Dermatology,
Karolinska Institutet, Stockholm, Sweden
Joke A.Bouwstra, Ph.D. Amsterdam Center for Drug Research, Leiden
University, Leiden, The Netherlands
David de Berker, B.A., M.B.B.S, M.R.C.P. Bristol Dermatology Centre,
Bristol Royal Infirmary, Bristol, United Kingdom
Sven Engström, Ph.D.* Department of Pharmacy, Uppsala University,
Uppsala, Sweden
Bo Forslind, M.D., Ph.D.† Department of Medical Biophysics, Karolinska
Institutet, Stockholm, Sweden
Ya-Wei Hsueh, Ph.D. Department of Physics, Simon Fraser University,
Vancouver, British Columbia, Canada
Leslie N.Jones, Ph.D. CSIRO Textile and Fibre Technology, Belmont,
Victoria, Australia
Neil Kitson, M.D., Ph.D., F.R.C.P.C. Division of Dermatology, Department
of Medicine, University of British Columbia, Vancouver, British Columbia,
Canada
Magnus Lindberg, M.D., Ph.D. Occupational and Environmental Medicine,
Stockholm Center of Public Health, and Occupational and Environmental
Dermatology, Karolinska Institutet, Stockholm, Sweden
Andrew J.G.McDonagh, M.B., Ch.B., F.R.C.P. Department of Dermatology,
Royal Hallamshire Hospital, University of Sheffield, Sheffield, United
Kingdom
Gopinathan K.Menon, Ph.D. California Academy of Sciences, San
Francisco, California, U.S.A.
*Current affiliation: Chalmers University of Technology, Göteborg, Sweden.

†Deceased.
xiv
Andrew G.Messenger, M.D., F.R.C.P. Department of Dermatology, Royal

Hallamshire Hospital, University of Sheffield, Sheffield, United Kingdom
David J.Moore, Ph.D. Unilever Research US, Edgewater, New Jersey,
U.S.A.
Lars Norlén, M.D., Ph.D. Department of Physics, University of Geneva,
Geneva, Switzerland
Gonneke S.K.Pilgram, Ph.D. Center for Electron Microscopy, Leiden
University Medical Center, Leiden, The Netherlands
Valerie Anne Randall, Ph.D. Department of Biomedical Sciences, University
of Bradford, Bradford, United Kingdom
Emma Sparr, Ph.D.* Department of Pharmacy, Uppsala University,
Uppsala, Sweden
Rachid Tazi-Ahnini, Ph.D. Division of Genomic Medicine, University of
Sheffield, Sheffield, United Kingdom
Jenifer Thewalt, Ph.D. Department of Physics and Institute of Molecular
Biology and Biochemistry, Simon Fraser University, Vancouver, British
Columbia, Canada
Desmond J.Tobin, Ph.D. Department of Biomedical Sciences, University of
Bradford, Bradford, United Kingdom
Philip Wertz, Ph.D. Dows Institute, University of lowa, lowa City, lowa,
U.S.A.
Carolyn Willis, Ph.D. Department of Dermatology, Amersham Hospital,
Amersham, United Kingdom
*Current affiliation: Utrecht University, Utrecht, The Netherlands.

1
An Introduction to the Book
Bo Forslind†
Magnus Lindberg
Stockholm Center of Public Health and Karolinska Institutet,
Stockholm, Sweden
Research is to a certain extent a victim of current trends. In dermatological
sciences, the decade of the 1970s was dominated by the rapidly evolving
immunological techniques. The last two decades of the twentieth century saw an
expansion of molecular techniques and genetic tools as the main instruments.
Lost in the wealth of research weapons was a global interpretation of research
data, an assessment of the relation between the new facts and the structure-
function relationships of the organs and organisms studied.
The present text is intended to fill some of this knowledge gap. The editors
have been fortunate enough to acquire chapters from young scientists who
already are at the forefront of research in their fields.
With a structure-function approach in mind, the chapters in this book give a
short overview of the techniques used by the various authors, with references to
standard textbooks on the subject. The intention is that the reader should be able
to understand how the results are obtained to validate them with respect to the
properties of the analytical equipment and techniques described.
The integument and its appendices show a number of similarities at microscopic
resolutions and also in their biochemical and immunological aspects, but there
are as well some conspicuous differences related to the function of respective
structures. This introduction highlights some of these properties at the same time
as it presents some of the content of the book.
1
KERATINIZATION
Keratinization is the rather diffuse denominator for the result of a differentiation
process that occurs in the integument and its appendices. Rather unsubstantiated
characterizations such as hard keratins and soft keratins appear especially in
older literature. Here we relate keratinization to the sulfur content, which varies
substantially. Functionally it has been shown that high sulfur content is a
characteristic of the keratin of nails, hooves, quills and so on, whereas the skin
†Deceased.
2 FORSLIND AND LINDBERG
represents the soft keratin and contains comparatively low amounts of sulfur. At
a macromolecular level, the low sulfur, high molecular weight proteins represent
the fibrous component, whereas the high sulfur protein fractions represent low
molecular weight proteins.
1.1
Keratinized Tissues
The integument and the appendices are what we name keratinized tissues; that is,
the main component of the fully differentiated tissue is a fibrous intermediate
filament protein structure, characterized by an unusually high content of sulfur
present in the amino acid cysteine. Cysteine-sulfur links stabilize the protein
chains, making them mechanically as well as chemically highly resistant. For
biochemical characterizationes the keratins are usually dissolved with
thioglycollate, which opens up the disulfide bonds of the cysteine. Such a
treatment is also the basis for the “cold perms” used by hair-dressers.
Keratins thus constitute a group of intermediate filaments and show different
chemical and immunological properties related to their origin. The
electrophoretic patterns obtained from the dissolved proteins of these three
tissues indicate chemical differences, which in turn suggest different
immunological properties. The electrophoretic patterns also reveal that nail
keratin and epidermal keratin are more related to each other than to hair keratin.
1.2
The Keratinized Cell
In general terms the keratinized cell can be described as an agglomeration of
fibrous components enclosed in a protein envelope. The formation of a
keratinized cell is in many respects similar to the phenomenon of apoptosis,
sometimes also called programmed cell death. This implies that the cell content
is disintegrated when the cell has fulfilled its mission in the production of fibrous
and other proteins, as well as lipids, and the delimiting cell membrane has been
replaced by a protein envelope. In fact, all keratinized cells have had their cell
membrane transformed from a lipid structure into a protein envelope composed
of highly cross-linked proteins, the most important being involucrin, supported
by loricrin, filaggrin, and small proline-rich proteins. The fibrous keratin inside
the cell envelope constitutes an internal reinforcement that puts restrictions on
the cell form in dry and wet conditions; that is, it ensures a definite cell form.
The cell contacts also differ in the epidermis and its appendices in relation to
the particular needs of the tissue. Thus in the epidermis the cell contacts are
desmosomes, whereas a tight-junction-like type of contact predominates in hair.
This is also the case of the dorsal nail plate, whereas the ventral nail plate cells
are joined by desmosome-like structures.
INTRODUCTION 3
2
THE LIPIDS OF CELL SURFACES
The cornified cell surface of the epidermal cells is rendered lipophilic by cross-
linked -hydroxy ceramides packed closely enough to cover the entire
corneocyte protein envelope. These w-hydroxy ceramides can be removed by
alkaline hydrolysis, indicating ester linkage to the proteins rather than amide
linkage. A component of the corneocyte protein envelope, involucrin, which has
about 20% of glutamate residues, is the likely candidate molecule for the
attachment of the ceramides.
It is interesting that the cuticular surface of the hair is studded in a similar
manner with lipids. In this case the lipids are saturated straight-chain and
branched-chain (18-methyleicosanoic acid) fatty acids. The fatty acids are
attached to the proteins through thioester bonds. It can be speculated that the
lipophilic surface of the cuticle cells promotes the spreading of sebum over the
hair surface, diminishing the risk of hair fiber tangles by “oiling” the surface.
3
THE HAIR
In the hair the cornified cells, the cortex cells, are elongated, spindlelike
structures with the keratin essentially oriented in the tip-tail direction. The
cortical cells are bonded to each other completely all along their surfaces with a
protein “glue,” the actual detailed composition of which still escapes us. It is,
however, likely to contain a lot of sulfur, and there are indications that this
substance has rubberlike properties. Further mechanical strength is provided by
the coupling between consecutive cells in the vertical direction. One interesting
aspect of hair fiber formation is that the inner root sheath is consolidated deep
down in the follicle. The follicle is also featured with a kind of a “corset” of
circularly arranged collagen fibers, which prevents sideways expansion due to
the growth pressure of the new cells formed at the matrix level. The rigidity of
the funnel formed by the inner root sheath and the surrounding connective tissue
causes the cells produced by cell division at the matrix level to become elongated
axially as they are forced (squeezed) trough the funnel structure. Hence the
spindlelike form of the cortex cells.
The keratin filaments of a cortex cell are completely confined to the
compartment surrounded by the protein “capsule” that forms the cell envelope.
Thus, the cortex cells are formed by a “programmed cell death” analogous to
that of the corneocytes of the epidermis. The filaments form a superstructure that
can be visualized as a helical arrangement of basic filaments, 8 nm wide. This
helical arrangement from the molecular to the macromolecular levels enhances
the mechanical strength of the composite structure.
We want the hair fibers of the scalp to be elastic, pliable. They show such
properties because the hair fibers mostly are more or less solid rods, sometimes
carrying a discontinuous medulla. In contrast, the eyelashes, being fibers that

have a prominent central medulla, are rather stiff structures. However, the tip of
an eyelash is short and solid and shows the property of being somewhat pliable,
which is why the eyelashes can be formed cosmetically with the aid of eyelash
curlers and/or mascara. Consider the limpness of a flat sheet of paper and notice
the change of properties (i.e., mechanical stability) when we form a half-cylinder
of the same sheet. This indicates why eyelashes have conspicuous medullae.
Hair growth, and even more so the lack of hair growth, is often presented in
tabloids and weekly magazines as a topic of particular interest. It may seem that
the cosmetic aspect of hair is dominating. A structure-function approach to the
study of hair growth from macroscopic to submicroscopic dimensions is
uniquely rewarding. Relationships between cellular organization, fibrillar
organization, and the remarkable mechanical properties of a hair fiber can be
understood from such a study.
The hair surface has been the object of study only recently, such interesting
findings as the presence of covalently bound lipids studding the cuticle cell
surface, making it highly hydrophobic not only are important when one is
comparing stratum corneum and hair but also have major implications for the
maintenance of hair structure and quality in a cosmetological sense. Again, the
psychological impact of an attractive hairdo influences individuals’ mental and
physiological health.
Male hormones, androgens, have important implications for hair growth.
Animal models in which this can be studied have highlighted important aspects
of the effects of androgen on hair growth. Seasonal variations quite evident in
animals are often so subtle in humans that we miss the effects unless we study
them carefully.
Male patterns baldness, which is related to androgens, is not a disease but a
physiological condition. Alopecia areata and related conditions, on the other
hand, attract attention by the medical establishment. Although we have an arsenal
of more or less successful treatments, we still lack a fundamental understanding
of the cause(s) behind the disease.
4
THE NAIL PLATE
Anatomically the nail plate can be dived into a dorsal (top) nail plate and a
ventral nail plate. The latter rests on the nail bed, which is dragged forward
passively by the continuously growing nail plate. The nail cells of the dorsal nail
plate are very flat cells, dimensionally similar to stratum corneum cells. The
dorsal nail plate cells are “glued” to each other along the entire periphery to form
a stiff sheet. Deeper down in the ventral nail plate the cell contour is tortuous and
the contact points are comparatively sparse, allowing the tissue a great deal of
elasticity and flexibility.
INTRODUCTION 5
The nail surface is smooth, sometimes even glossy. A scanning electron

microscope view of the surface reveals that the dorsal nail plate cells interlock
like a jigsaw puzzle. The dorsal nail plate cells are so flat because they are
“squeezed” into this form by a collagen tunnel inserting on the tip of the
phalanx. The cells of the ventral nail plate do not suffer this “squeeze” because
they originate in front of the collagen tunnel.
Fibrillar orientation in the nail varies with depth in the nail matrix. The dorsal
nail plate cells have a keratin filament orientation similar to that of the stratum
corneum cells. In contrast, the keratin filaments in the ventral nail cell are highly
ordered perpendicularly to the growth axis, preventing forces along the growth
axis from damaging the nail plate down to the matrix level. This orientation
allows the nail to bend. The anatomical arrangement of the cells in the dorsal and
the ventral nail plate, together with the fibrillar arrangement, provides a stiff
dorsal nail plate, which forms a sharp instrument with which we can peel
oranges and bananas. The function of the ventral nail plate is to distribute loads
imposed on the dorsal nail plate in such situations.
The mechanical properties also are a function of tissue organization. The
stratum corneum is a flat sheet, flexible and soft. In contrast we experience the
nail as “hard.” In reality the word to be used is “stiff”, since the physical term
“hard” is more applicable to diamonds, stainless steel, and marble.* In addition
to the cellular arrangement, the nail plate often shows two curvatures, a
longitudinal curve with a very large radius and a curvature at right angles to this
with a shorter radius, especially in women who have a slender finger skeleton.
Comparing the limpness of a flat sheet of paper and the change of properties
when we form a half-cylinder of the same sheet provides an idea of the
robustness of nail construction.
Nails correspond to claws in mammals. The nail is another structure that reveals
a number of properties that can be understood from a structure-function
perspective. Such information is available through the application of an arsenal of
biophysical methods and approaches, and from knowledge of the morphogenesis
and differentiation of the nail, we can describe some of the dynamics of nail
formation.
5
THE THIN VEIL OF THE SKIN BARRIER
A close look at the anatomy of the skin teaches us that the cellular epidermis that
forms the watertight barrier toward the environment is an extremely thin film.
*Given that the physical meaning of “hard” refers to properties of complete elasticity
(e.g., a steel ball bouncing on a marble floor loses only a minute fraction of its kinetic
energy at the perfect elastic collision with the marble), the concept of hardness in a
biological sense is different from that of the physical one. “Hard” in a biological sense can
therefore be interpreted as “hard to bend, hard to break.”
The entire thickness of the cellular epidermis is about 0.1 to 1.0 mm, and the
stratum corneum, where the barrier resides, is about 0.01 mm thick. This means
that our watertight cover has a thickness that is less than the cellophane wrap
that covers your luncheon sandwiches.
In the normal human skin the corneocytes of stratum corneum are extremely
flat structures with a widths of about 30 µm and a thickness of 0.3 µm. They are
joined to each other by numerous protein “rivets,” corneodesmosomes. The cell
contacts also differ in the epidermis and its appendices in relation to the
particular needs of the tissue. The corneocytes function as a scaffold to
mechanically protect the lipids deposited in the extracellular space. It can be
intuitively understood that the lipid bilayer structures that form the skin barrier
have minimal capacity to withstand mechanical forces tending to shear, or even
disrupt, these two-dimensionally ordered structures. Thus, a mechanically
resistant cover is mandatory if the barrier function is to remain intact under normal
conditions. Since the skin barrier always must be intact, continuous renewal of
the barrier and consequent shedding of the surface layer must take place to keep
the horny layer thickness constant. At the surface, where the cells eventually are
freed in groups of two or more, the “rivets” have been enzymatically digested to
free them from their neighbors. This enzymatic attack is monitored by changes in
the water gradient over the stratum corneum. In the narrow box of a skin
corneocyte, the keratin filaments are “randomly” distributed in the plane of the
cell and provide inside reinforcement. This construction allows the cell to swell
only 1 to 2 % in the plane of the cell but up to 25 % in a vertical dimension. We
can see that this prevents the skin surface of the body from roughening when
moist, thus diminishing the risk that the thin surface film will be torn on contact
with the environment.
The barrier function of human skin has attracted deep interest from
pharmacologists, who view the skin as a medium for transport. Transport
interests are also evident in occupational dermatology. Most such investigations
can be thought of as “black box” studies in which the penetration rates of a
substance and of its possible metabolites are recorded after passage through the
skin. Such facts have been the substance of theoretical/mathematical models for
skin barrier transport. When transport has been increased, investigators have
attributed to the enhancement effect “pores.” However, no substantial, molecular
models for the barrier or such pores were presented until the last decade of the
past century. An attempt to give a molecular basis for skin barrier structure and
function was presented with the “domain mosaic model” in 1994. In the ensuing
years this model has been criticized but also developed, and it remains an
inspiration (or irritation) to researchers in the skin barrier field. Thus, the “black
box” theories are complemented with perceptions and ideas based on molecular
considerations. Three-dimensional, cubiclike organization of the lipids forming
the barrier has evolved as a plausible idea, since the transformation from a cubic
to a lamellar structure may require only minimal energy; it also may be faster
INTRODUCTION 7
and may imply increased process control compared with the diffusion and fusion
processes proposed by electron microscopists.
A general knowledge of long-chain lipid behavior provides a basis for
understanding the structure and function of the human skin barrier. In addition,
the principle behind phase diagrams and the results of atomic force microscopy
will aid us in the process of developing such an understanding.
Modern biophysical techniques such as Fourier transform infrared
spectroscopy and nuclear magnetic resonance studies on experimental systems
mimicking the composition of the human skin barrier have given us a new
understanding of the complexity of lipid organization. These techniques allow us
to understand the effects on barrier structure of variations in the composition of
the bilayer-forming lipids. Such information will eventually help us to monitor
the (genetic) repair of defective barriers or the penetration rates for topically
applied pharmacological substances aiming at local or systemic action.
X-ray diffraction and electron diffraction have been specialized tools in the
study of lipid structure arrangements in the stratum corneum. The diffraction
techniques allow the description of ordered systems.
Lipid analysis is consequently a most interesting topic. The complexity of the
composition of barrier lipids was outlined in the 1970s with the work by Gray
and Yardley. The ceramides, unique lipids for the human organism, have been in
focus for almost two decades, both from a scientific viewpoint but also as a
“weapon” in the competition among cosmetic manufacturers. It has been
suggested that lack, or diminished proportions, of certain ceramides is
responsible for the barrier defect of atopic dermatitis.
This proposal has not been studied completely, and the possibility remains
that more generalized factors such as differences in the proportions of barrier
components may be more important than the absolute amounts of certain lipid
species. A development from thin-layer chromatography via HPLC-LSD (high-
performance liquid chromatography with light-scattering detector) toward mass
spectrometry will no doubt provide more reliable data than we have had in the
past.
The lamellar bodies seen in the electron microscope have a history that goes
back to the 1960s when Gedeon Matoltsy described membrane coating granules
(MCG) in a skin section prepared for transmission electron microscopy. The
MCGs have since been named lamellar bodies, and electron micrographs have
suggested that their content is extruded into the extracellular spaces between the
stratum granulosum cells and the first stratum corneum cells. There the sheets of
lamell have been suggested to fuse into a continuous bilamellar structure. This
interpretation is a purely morphological one and ignores the energy-consuming
nature of the processes involved and the absence of any evidence for small lipid
moities that can close the ends of the lamellar sheet, preventing exposure of the
hydrophobic chains to the tissue-water environment. Alternative ways of
explaining the formation of the organized lipid bilayer structure of the barrier
have recently been presented and are discussed in this volume.
Seldom are comparative aspects discussed in dermatological science.

Comparative morphology at electron microscopic resolution gives us an insight
into cellular and subcellular organization of the integument. Such structural
aspects inevitably underline the uniqueness of the human integument but also
provide insights of structure-function relationships that increase our ability to
interpret data from the human integument.
An understanding of the irritative reaction and barrier changes in skin diseases
provides insights into the mechanisms that precede the development of a contact
allergic reaction and suggests how penetration into and through the skin can be
altered. Clinical data and experimental data form the basis for an understanding
of what is going on the human skin in contact with noxious substances.
6
SUMMARY
The similarities we observe are that the fully differentiated cells of the
integument—skin, hair, and nail—are the result of programmed cell death,
giving as a final product a protein-bounded compartment containing highly
hydrophilic intracellular fibrous keratin, which provides internal reinforcement
for cells of the skin and its appendices.
Table 1 lists the similarities and differences that have been noted between
fully differentiated integumental cells of skin, hair, and nails.
It is also obvious that a structure-function perspective increases our possibility
to understand both normal and pathological processes in the skin and its
appendages. The intention of this book is to provide data in this field of research.
TABLE 1 Comparison of Skin, Hair, and Nail Cells and Stratum Corneum Cells
Similarities Differences
The fully differentiated cells of the There are minor differences in the
integument (skin, hair and nail) are the chemical and immunological properties of
result of a programmed cell death giving skin, hair, and nail cells.
as a final product a protein-bound
compartment containing highly
hydrophilic intracellular fibrous keratin
that provides internal reinforcement for
cells of the skin and its appendices
Hair and nail cells are shaped by Stratum corneum cells suffer no
restrictions imposed by the shape of their mechanical restriction at formation.
anatomical site.
Hair and dorsal nail plate cells are joined Stratum corneum cells are joined by
by an intercellular protein glue along the corneodesmosomes, which allow the cells
entire periphery. Cells are lost by wear and to be shed eventually, after an enzymatic
tear. attack on these structures.
2
Structure and Function of the Skin Barrier: An
Introduction
Bo Forslind†
Magnus Lindberg
Stoc kholm Center of Public Health and Karolinska Institutet,
Stockholm, Sweden
The skin provides the barrier between the environment and the internal milieu.
Maintaining body water (i.e., the water homeostasis) is vital for normal
physiology [1, 2]. In this respect, regulation of body water volume and
composition is a function of the kidneys, and the integument should provide a
water-impermeable barrier to minimize transepidermal water loss. There is,
however, a small, very constant loss of water across the skin calledperspiratio
insensibilis, providing the water needed as a plasticizer for the keratin in the
corneocytes to maintain their function. Today it is acknowledged that the water
diffusion barrier is located in the outermost part of epidermis, the stratum
corneum [3, 4], and that its function depends on the content and structure of the
intercellular lipids [5, 6]. Producing and maintaining the water diffusion barrier
is a dynamic process [7] that depends on the status and function of the skin (e.g.,
inherited disorders of keratinization, other concomitant skin diseases, generalized
internal diseases) and influences from factors in the external environment.
The structure, function, and homeostatic control mechanisms of the barrier are
of interest not only to those engaged in basic dermatological research. In recent
years we have seen an increasing interest in techniques for transepidermal
(transdermal) drug delivery systems. Barrier properties are also of interest with
respect to the application of pharmaceutical treatments to the skin (e.g.,
moisturizers) and in cosmetology. It has become evident, as well, that the stratum
corneum is a target organ for new treatment strategies for inflammatory skin
diseases [8]. Finally, but not least, skin barrier and function are of central
importance for those engaged in occupational dermatology and medicine,
working on skin exposure, penetration, and contact dermatitis. This chapter
presents an overview of the structure and function of the epidermis in relation to
barrier properties as an introduction to the subsequent chapters on the skin in this
book.
†Deceased.
1
EPIDERMAL ARCHITECTURE AND FUNCTION
Epidermis is a stratified epithelium separated from dermis by the basal
membrane (c f. Refs. [9, 10]). Normal adult epidermis is composed of three major
cell populations: keratinocytes, immune-competent dendritic Langerhans cells,
and melanin-producing melanocytes. Keratinocytes constitute approximately
95% of the cell volume and contain the main structural protein, keratin [11].
They are responsible for producing and maintaining the stratum corneum, the
diffusion barrier [9]. Among other functions keratinocytes also participate in
inflammatory and immunological processes and provide the local micromilieu for
such events [12, 13].
Based on morphology as seen with light and electron microscopy [10, 14], the
epidermis has conventionally been divided into five different cells layers
(Fig. 1): the one-cell-thick basal cell layer (stratum basale or stratum
germinativum), the several-cells-thick spinous cell layer (stratum spinosum), the
granular cell layer (stratum granulosum) with several cells, the transition cell
layer (one cell), and the cornified cell layer (stratum corneum) containing 10 to
30 cell layers.
Epidermal stem cells are located in the basal layer [15, 16]. Following cell
division, one cell migrates toward the skin surface with simultaneous maturation
and differentiation to form the corneocytes of the stratum corneum. During this
differentiation the keratinocytes change their form from cuboidal in the basal
layer to flat, hexagonal corneocytes covering a large surface area in stratum
corneum (Fig. 2). The differentiation is also associated with changes in the
content of intracellular organelles, lipid metabolism, keratin expression, and the
elemental content of the cells [9–11, 17, 18]. In the upper part of the spinous
layer, cytoplasmic organelles called lamellar bodies (membrane-coating granules,
Odland bodies, lamellar granules) [19–21] can be found. They contain preformed
stratum corneum lipids and enzymes. Lamellar bodies are formed in the
endoplasmic reticulum, and through exocytosis their content is expelled into the
intercellular space in the transition from stratum granulosum to stratum corneum
[22–24]. Recently [25] the structure, nature, and dynamics of the lamellar bodies
have been disputed, and it was suggested that these organelles represent a
continuum between the endoplasmic reticulum and the intercellular space rather
than discrete organelles. Stratum granulosum is named from the appearance in
the cytoplasm of dense granules, keratohyaline granules [9, 10]. These
keratohyaline granules, the precursor of the amorphous matrix of the
corneocytes, consist mainly of the histidine-rich protein filaggrin. In the
transition from stratum granulosum to corneum, the cells lose their nuclei and
cell organelles and achieve a thickened cell membrane. Throughout the
epidermis, the cells are connected to one another by specialized membrane
structures, the desmosomes. The basal surfaces of basal cells are connected to the
basal membrane through hemidesmosomes.
STRUCTURE AND FUNCTION OF THE SKIN BARRIER 11
FIGURE 1 Schematic presentation of a vertical section through epidermis.

2
THE FORMATION OF THE HUMAN SKIN BARRIER
Normally the stratum corneum, the diffusion barrier, is 10 to 30 cell layers thick,
corresponding to approximately 5 to 20 µm. It can be depicted as flat, protein-
rich hexagonal corneocytes embedded in a lipid-rich intercellular space [26, 27].
In the transition from stratum granulosum, the volume of the intercellular space
is expanded from a few percent to approximately 15% of the volume in stratum
corneum. To meet the demands of an intact and functional diffusion barrier, there
is continuous renewal of cells and lipids from the viable epidermis and
continuous shedding of corneocytes from the skin surface to ensure a constant
thickness of the horny layer [28]. Under normal conditions, cell renewal,
migration, differentiation, and shedding of keratinocytes/corneocytes occur in an
orderly manner, regulated by a number of control mechanisms. It has been
demonstrated that the transepidermal water flow is of importance for the
homeostasis of the barrier and that also changes in the elemental profiles across
epidermis (e.g., calcium concentrations) are crucial homeostatic signals [7].
The barrier is composed of corneocytes with a thickened cell membrane and
intercellular lipids, some of which are bound to the cell membrane [29–36]. The
thickened cell membrane is called the cornified envelope and today it is known
FIGURE 2 A schematic drawing of the suggested epidermal proliferative unit. The

relation between cells in stratum basale (the proliferating cell compartment) and a fully
differentiated corneocyte in stratum corneum is shown. Cell proliferation is controlled and
regulated within a defined proliferative unit (cell-to-cell communication indicated with
arrows) to provide the smooth skin surface and a functional barrier.
to consist of two parts: an inner protein envelope and an outer lipid part.
Corneocytes still express desmosome rivets, so-called corneodesmosomes [37]
(Fig. 3). From a functional point of view, the corneocytes with their
corneodesmosomes present a rigid, stable structure that can act us a scaffold for
the barrier lipids [38].
2.1
Corneocyte Structure
A corneocyte is flat, about 30 µm in diameter and approximately 0.3 µm thick. It
contains keratin fibrils, 8 µm in diameter, in an amorphous matrix [11]. The
fibrils are horizontally oriented and span the inside of each corneocyte.
Corneocytes can bind substantial amounts of water, but the arrangement of the
fibrils prevents horizontal swelling; the cell can increase more than 25%
vertically, however [39]. This minimizes the risk of surface breaks when
mechanical stress is imposed on wet skin.
FIGURE 3 The arrangement of cells (corneocytes) within stratum corneum. The cells are
still connected through specialized desmosomes. These desmosomes are dissolved
enzymatically in stratum corneum before shedding of the cells. The intercellular space
between the corneocytes is filled with lipids. Within the corneocytes the keratin filaments
are arranged horizontally (x-z plane) and also bound to the inner part of the cell
membrane, providing resistance against tension (or swelling) in this plane.
The corneocyte envelope consists of two parts: a thicker protein envelope (~15
nm) composed of cross-linked structural proteins adjacent to the interior
cytoplasm and a thinner (~4 nm) lipid envelope on the intercellular face of the
protein. In the inner protein membrane the proteins (e.g. filaggrin, involucrin,
elafin, loricrin, keratin intermediate filaments) are crosslinked by disulfide and -
( -glutamyl) lysine isopeptide bonds. This provides a very stable structure. The
outer lipid envelope consists of ceramides ( -hydroxy ceramides) linked to
involucrin in the protein part of the envelope.
2.2
Barrier Lipids
The intercellular lipids of the stratum corneum are derived from lipids processed
in the viable epidermis and then extruded into the intercellular space. Although
stratum corneum formerly was considered to be metabolic inactive, it hosts a

continuous processing of both lipids and proteins, and several enzymes
responsible for this have been identified [40–42]. The lipid composition of
stratum corneum is unique and is a mixture of mainly different ceramides, free
fatty acids, sterols, and cholesteryl esters; a substantial part of the ceramides and
free fatty acids have long carbon chains (C>20.0). It has been demonstrated that
the long-chain ceramides of stratum corneum, in the presence of cholesterol and
fatty acids at physiological pH, do have the same capacity to form bilayers as
phospholipids. The intercellular lipids in stratum corneum are arranged in a
bilamellar fashion containing both polar and nonpolar regions [29, 31]. It is
proposed that the lipid structure spans the distance between the cornified
envelopes of adjacent corneocytes and that the lipids are anchored through
binding to the -hydroxy ceramides in the lipid envelope [31–36].
3
PROPERTIES OF A LIPID MEMBRANE OR BARRIER
The physical state of the lipids determines the properties of a lipid membrane or
barrier. Lipids that form biological membranes are characterized by a
hydrophilic head group and a hydrophobic part, usually a carbon chain. From
physical, thermodynamic considerations it can be shown that much energy is
required to keep the hydrophobic part of a lipid dissolved in a water solution.
Thus, the lipids tend to aggregate in micelles or bilayer structures. The factors
that determine how stable such aggregates are include temperature, the length of
the hydrophobic carbon chain, the degree of unsaturation (double bonds), the
amount of cholesterol, the presence of divalent ions, and pH. It has been
postulated (c f. the domain mosaic model described in Ref. [38]) that a major
part of the lipids in the intercellular space of stratum corneum are packed in a
crystalline phase, that is, a membrane with little or no mobility, which hinders
water diffusion. There will also be a smaller fraction of lipids in a liquid
crystalline phase, with intermembrane movements that permit water diffusion.
This lipid arrangement would be compatible with a physiological function of an
adaptable diffusion barrier. The model for lipid structure and lipid arrangement
has recently been further explored by several investigators, including Norlén
[43] and Bouwstra and coworkers [29].
4
BARRIER MODELS
In the mid-1970s Michaels et al. presented the “bricks and mortar” stratum
corneum architecture, implicating a two-compartment system with a protein
phase (corneocytes) in a lipid phase (intercellular lipids) model [26] (Fig. 4).
This concept was further developed by Elias and coworkers and formed the base
for their pioneering work on the skin barrier [27]. The “bricks and mortar”
model is not a structural but more a conceptual model of the barrier, in as much
as it does not fully address the structure-function relation. In 1994 Forslind [38]
proposed a model for the diffusion barrier of the stratum corneum called the
“domain mosaic model.” According to this model, which is based more on
structure-function consideration than that of Elias, most stratum corneum lipids
are arranged in domains with a crystalline packing, minimizing penetration of
water. The crystalline domains have fringes with lipids in a fluid crystalline
phase, permitting diffusion of water.
The domain mosaic model has recently been further explored by Norlén, who
suggests that the lipids in the basal part of the stratum corneum are arranged in a
single gel phase [43]. This is proposed to be most optimal structural arrangement
from both functional and energy (thermodynamic) point of view. The single gel
phase has been disputed by Bouwstra and coworkers [29], who describe a
“sandwich model” permitting different lipidphases to be present.
FIGURE 4 An adaption of the brick-and-mortar model initially suggested by Michaels et al. [26]. The corneocytes can be seen as protein
loaded bricks with a mortar of intercellular lipids. Penetration through the skin is mainly through the intercellular space.
5
IMPLICATIONS FOR PENETRATION THROUGH THE
SKIN BARRIER
A structural arrangement of the stratum corneum, with protein-rich corneocytes
surrounded by a lipid-rich intercellular space in which the lipids are arranged in
bilayers with hydrophobic and hydrophilic parts and variable forms of membrane
packing, has implications for penetration through the barrier. Today, there is a
consensus about penetration pathways through the skin barrier. Under normal
conditions, there is no penetration through the corneocytes. They are essentially
permeable only to water. This implies that both hydrophobic and hydrophilic
substances penetrate via the lipids in the intercellular space. Water can probably
be found between the lipid bilayers, forming a continuum that allows transport of
hydrophilic substances in the plane of the bilayers. It has also been shown that
hydrophobic substances have the capacity to diffuse in the plane of the bilayers.
This means that both hydrophilic and hydrophobic substances can be transported
in the plane of the bilayers [44]. To penetrate into and through stratum corneum,
however, there must be penetration across the bilayers, According to the domain
mosaic model, the bulk of the lipids in stratum corneum are arranged (packed) in
a crystalline phase that is virtually impermeable to water. These crystalline
domains are separated by domains of lipids arranged in a liquid crystalline
fashion, allowing penetration of water. The development of the single gel phase
model, or the sandwich model, further demonstrates the complexity of lipid
structure in stratum corneum.
The functional implication of the different models is that, although stratum
corneum is thin, the penetration pathway is long under normal conditions. To
penetrate from the outside to the inside a water molecule must find lipids in a
liquid crystalline phase. If skin is exposed to substances that interact with the
lipid phase (e.g. penetration enhancers, detergents, lipids), this can be expected
to influence penetration through the skin by changing the packing of the lipids.
6
CONCLUSIONS
During the past two decades we have seen enormous increases in our knowledge
about barrier structure and function, and the field is still expanding. It involves
not only basic researchers. The new insights into barrier structure and function
have implications for those in clinical dermatology, for researchers developing
new treatment modalities, and for those engaged in occupational and
environmental dermatology and medicine. Just recently we have seen an
evolving interest in the field of skin exposure, penetration, and possible systemic
and local effects. For some toxic substances (e.g., pesticides) the skin can be the
major route of entry into the human body. Based on present knowledge, we
believe that we, today, are standing in the doorway to new, exciting challenges in
the work related to the skin barrier and its structure and function. The chapters that
follow offer a more extensive and detailed description and discussion on lipids,
lipid structure and function, stratum corneum models, and mechanisms and
effects of pathological changes in the skin barrier function.
ACKNOWLEDGMENTS
Figures 1 to 3 were produced by Mr. Kalle Forss.
REFERENCES
1. Forslind B. The skin: upholder of physiological homeostasis. A physiological and
(bio)physical study program. Thromb Res 1995; 80:1–22.
2. Forslind B. The structure of the human skin barrier. In: L Kanerva, P Elsner, JE
Wahlberg, HI Maibach, eds. Handbook of Occupational Dermatology, Berlin,
Springer-Verlag; 80:2000:56–63.
3. Elias PM, Friend DS (1975) The permeability barrier in mammalian epidermis.
J.Cell. Biol. 65:180–191.
4. Landmann L. (1988) The epidermal permeability barrier. Anat. Embryol. 178: 1–
13.
5. Wertz PW, van den Bergh B. The physical, chemical and functional properties of
lipids in the skin and other biological barriers. Chem Phys Lipids 1998; 91: 85–96.
6. Potts RO, Francoeur ML (1991) The influence of stratum corneum morphology on
water permeability. J. Invest. Dermatol. 96(4):495–499.
7. Feingold KR, Elias PM. The environmental interface: regulation of permeability
barrier homeostasis. In: M.Loden & H.I.Maibach, eds. Dry Skin and Moisturizers.
Chemistry and Function. Boca Raton, FL: CRC Press, 2000: 45–58.
8. Elias PM, Feingold KR. Does the tail wag the dog? Role of the barrier in the
pathogenesis of inflammatory dermatoses and therapeutic implications. Arch
Dermatol 2001; 137:1079–1081.
9. Eady RAJ, Leigh IM, Pope FM. Anatomy and organization of human skin. In: RH
Champion, JL Burton, DA Burns, SM Breathnach, eds. Rook/Wilkinson/Ebling/
Textbook of Dermatology Vol 1, 6th. Oxford: Black-well Science, 1998:37–57.
10. Odland GF. Structure of the skin. In: LA Goldsmith, Physiology, Biochemistry,
and Molecular Biology of the Skin, New York: Oxford University Press, 1991:3–
62.
11. Steinert PM, Freedberg IM.Epidermal structural proteins. L.A.Goldsmith
Physiology, Biochemistry, and Molecular Biology of the Skin. 2nd ed, New York:
Oxford University Press, 1991:113–147.
12. Elias PM, Wood KC, Feingold KR. Epidermal pathogenesis of inflammatory
dermatoses. Am J Contact Dermatitis 1999; 10:119–126.
13. Nickoloff BJ. Immunological reactions triggered during irritant contact dermatitis.
Am J Contact Dermatitis 1998; 9:107–110.
14. Breathnach AS. Aspects of epidermal ultrastructure. J Invest Dermatol 1975; 65:2–
15.
15. Lavker RM, Sun TT. Epidermal stem cells. J Invest Dermatol. 81:1983; (suppl):
121S-127S.
16. Dover R. Epidermal kinetics and stem cells. In: eds. I.M.Leigh, E.B.Lane,
F.M.Watt. The Keratinocyte Handbook., Cambridge: Cambridge University Press,
1995.
17. von Zglinicki T, Lindberg M, Roomans GM, Forslind B (1993) Water and
iondistribution profiles in human skin. Acta Dermatol. Venereol. (Stockh.) 73: 340–
343.
18. Warner RR, Myers MC, Taylor DA. (1988) Electron probe analysis of human skin:
determination of the water concentration profile. J. Invest. Dermatol. 90: 218–224.
19. Odland GP, Holbrook K. (1981) The lamellar granules of epidermis. Curr. Problems
Dermatol. 9:29–49.
20. Matoltsy AG. (1966) Membrane coating granules of the epidermis. J. Ultrastruct.
Res. 15:510–515.
21. Lavker RM. (1976) Membrane coating granules: the fate of the discharged
lamellae. J. Ultrastruct. Res. 55:79–86.
22. Landmann L. Epidermal permeability barrier. Transformation of lamellar granule-
disks into intercellular sheets by a membrane fusion process, a freeze fracture
study. J Invest Dermatol 1986; 87:202–209.
23. Elias PM, Cullander C, Mauro T, Rassner U, Kömüves, Brown BE, Menon GK.
(1998) The secretory granular cell: the outermost granular cell as a specialized
secretory cell. J. Invest. Dermatol. Symp. Proc. 235–236.
24. Madison KC, Sando GN, Howard EJ, True CA, Gilbert D, Swartzendruber DC,
Wertz PW. (1998) Lamellar granule biogenesis: a role for ceramide
glycosyltransferase, lysosomal enzyme transport, and the Golgi. J. Invest.
Dermatol. Symp. Proc. 3(2):80–86.
25. Norlén L. (2001) Skin barrier formation: the membrane folding model. J. Invest.
Dermatol. 117(4):823–829.
26. Michaels AS, Chandrasekaran SK, Shaw JE. (1975) Drug permeation through
human skin: theory and in vitro experimental measurements. AIChE J. 21(5):985–
996.
27. Elias PM. Lipids and the permeability barrier. Arch Dermatol Res 1981; 270: 95–
117.1982.
28. Egelrud T.Desquamation in the stratum corneum. Acta Dermatol Venereol Supp
2000; 208:44–45.
29. Bouwstra JA, Dubbelaar FER, Gooris GS, Ponec M. The lipid organisation in the
skin barrier. Acta Dermatol Venereol. suppl. 2000; 208:23–30.
30. Chang F, Swartzendruber DC, Wertz PW, Squier CA. (1993) Covalently bound
lipids in keratinizing epithelia. Biochim. Biophys. Acta 1150: 98–102.
31. Downing DT. (1992) Lipid and protein structures in the permeability barrier of
mammalian epidermis. J. Lipid Res. 33:301–313.
32. Swartzendruber DC, Wertz PW, Madison KC, Downing DT. Evidence that the
corneocyte has a chemically bound lipid envelope. J Invest Dermatol 1987; 88:709–
713.
33. Swartzendruber DC, Wertz PW, Kitko DJ, Madison KC, Downing DT. (1989)
Molecular models of the intercellular lipid lamellae in mammalian stratum
corneum. J. Invest. Dermatol. 92:251–257.
34. Wertz PW. The nature of the epidermal barrier: biochemical aspects. Adv Drug
Deliv Rev 1996; 18:283–294.
35. Wertz PW, Madison KC, Downing DT. (1989) Covalently bound lipids of human
stratum corneum. J. Invest. Dermatol. 91:109–111.
36. Wertz PW, Downing DT. (1987) Covalently bound -hydroxyacylsphingosine in
the stratum corneum. Biochim. Biophys. Acta 917:108–111.
37. Fartach M, Bassukas ID, Diepgen T.Structural relationship between epidermal lipid
lamellae, lamellar bodies and desmosomes in human epidermis: an ultrastructural
study. Br J Dermatol 1993, 128:1–9.
38. Forslind B (1994) A domain mosaic model of the skin barrier. Acta Dermatol
Venereol. (Stockh.) 74:1–6.
39. Norlén L, Emilson A, Forslind B. (1997) Stratum corneum swelling. Biophysical
and computer assisted quantitative assessments. Arch. Dermatol Res. 289:506–513.
40. Elias PM, Holleran WM, Calhoun DQ, Brown BE, Behne M, Feingold KR,
Permeability barrier homeostasis: the role of lipid processing. In: M.Loden & HI
Maibach eds. Dry Skin and Moisturizers. Chemistry and Function., Boca Raton, FL:
CRC Press, 2000:59–70.
41. Bonté F, Saunois A, Pinguet P, Meybeck A. Existence of a lipid gradient in the
upper stratum corneum and its possible biological significance. Arch. Dermatol
Res. 1997; 289:78–82.
42. Holleran WM, Takagi Y, Menon G, Legler G, Feingold KR, Elias PM. (1993)
Processing of epidermal glycosylceramides is required for optimal mammalian
cutaneous permeability barrier function. J. Clin. Invest. 91:1656–1664.
43. Norlén L. (2001) Skin barrier structure and function: the single gel-phase model. J.
Invest. Dermatol. 117(4):830–836.
44. Johnson ME, Blankschtein D, Langer R. (1997) Evaluation of solute permeation
through the stratum corneum: lateral bilayer diffusion as the primary transport
mechanism. J. Pharm. Sci. 86(10):1162–1172.
3
Lipid Phase Behavior: A Basis for an
Understanding of Membrane Structure and
Function
Emma Sparr* and Sven Engström†
Uppsala University, Uppsala, Sweden
1
THE STRUCTURE AND FUNCTION OF STRATUM
CORNEUM
The brick-and-mortar model of the outermost part of human skin, the stratum
corneum or “horny layer,” was introduced by Michaels et al. [1] and later
developed by Elias [2]. The model describes a tortuous structure, 2 to 10 µm
thick, in which corneocyte cells (80–90 vol %) are embedded in a lipid matrix,
which in turn consists of a complex mixture of different lipid species arranged in
stacked bilayers parallel to the skin surface (Fig. 1) [3]. The major lipid classes
are ceramides, cholesterol, and fatty acids (Fig. 2) [4]. Compared with other
biomembrane lipids, the skin lipids have relatively long hydrocarbon chains,
which make them crystalline at body temperature when isolated in test tubes and
submerged with water.
The role of stratum corneum is to protect the body from uncontrolled passage
of substances in both directions. The stacked lipid bilayer makes an optimal
protection in this respect, since the alternating polar and nonpolar domains of the
lipid bilayer matrix hinder very polar as well as very nonpolar substances in their
diffusion. Perhaps the most important task of stratum corneum is to prevent
water from diffusing out from the body to any great extent. However, stratum
corneum is not watertight, since it allows for a loss of about 100 to 150 mL per
day per square meter of skin surface, the so-called transepidermal water loss
(TEWL) [5]. Moreover, gases such as oxygen and carbon dioxide are expected to
pass through the skin to some extent [6]. Stratum corneum thus balance physical,
chemical, and biological demands, and its lipid constituent, the “mortar,” must
be scrutinized with this complex picture in mind.
Skin is attractive for drug therapy because it offers an easily accessible route
without liver first-pass metabolism. For transdermal applications (i.e., when the
*Current affiliation: Utrecht University, Utrecht, The Netherlands.

†Current affiliation: Chalmers University of Technology, Göteborg, Sweden.
22 SPARR AND ENGSTRÖM
FIGURE 1 Schematic drawings of the epidermis (from Ref. 3), the stratum corneum
bricks-and-mortar model, and the stacked bilayers of the stratum corneum extracellular
lipids.
drug must reach the systemic circulation), the protecting role of stratum corneum
must be overcome to reach an effective uptake. One strategy for overcoming the
barrier property entails the use of so-called chemical penetration enhancer, for
example, solvents (e.g., propylene glycol) and oleic acid. Physical forces such as
electrical voltage (iontophoresis, electroporation), ultrasound (sonophoresis), and
mechanical pressure are also used to overcome the skin barrier. In a delivery
situation, the number of drug molecules as well as solvent and/or enhancer
molecules typically exceeds by orders of magnitude the number of stratum
corneum lipids beneath the transdermal patch. In such cases it may be difficult to
distinguish between different transport mechanisms, which may be a complex
combination of dissolution of lipids, changes in lipid structure, formation of
solvent pools, and so on.
In 1994 Forslind presented the domain mosaic model (DMM), which
addresses several chemical and physical properties of stratum corneum [7]. In
view of the complex lipid composition, the DMM suggests a tortuous structure at
the lipid matrix level that takes in to account the preferred crystallinity of the
many long-chain lipids, which form mosaic pieces surrounded by “joints” in the
liquid state (Fig. 3). Besides giving inspiration for an understanding of the
transport properties of stratum corneum, the DMM gives an explanation for the
mechanical properties of skin. Important questions regarding the model are the
sizes and compositions of the mosaic pieces and the surrounding joints, and how
they are formed.
Other models of the lipid matrix of stratum corneum emphasize on the
molecular arrangements of the lipids [8, 9] more than the transport properties
these structures imply. A recent “single-phase” model proposes the existence of a
laterally homogeneous lipid matrix [10]. Taken together, these models
emphasize the importance of a deeper insight into the physicochemical
properties of complex lipid systems to promote understanding of the barrier
LIPID PHASE BEHAVIOR 23
FIGURE 2 Molecular structure of (a) fatty acid (palmitic acid, C16:0) in its undissociated
state, (b) cholesterol, (c) ceramide 3 (C16 CER 3), and (d) ceramide 1 (CER 1).
function of the stratum corneum. Irrespective of route—intracellular (through the
corneocytes) or intercellular—any molecule entering or leaving the body through
skin must pass the lipid matrix.
The section that follows provides introduction to the physical behavior of
lipids, with focus on stratum corneum lipids. The aim of the presentation is to
give the reader a general foundation that can be used as a tool for the
understanding of stratum corneum lipid structure and function. Sections 2 and 3
cover lipid phase behavior, including lipid structure, phase transitions, and
domain formation. The coupling between the lipid phase behavior and the barrier
properties of lipid membranes is treated in Section 4. The fact that skin separates
two regions with extremely different water contents may have a profound effect
FIGURE 3 Schematic illustration of the stratum corneum extracellular lipid bilayers,

where each lipid bilayer can be envisioned as a mosaic of crystalline domains held
together by lipids in a liquid crystalline state (the domain mosaic model) [7]. Diffusing
molecules will mainly choose the route in which resistance to diffusion is lowest (i.e., the
liquid crystalline domains), and the solid domains can be regarded as obstructions. (From
Ref. 3.)
on its phase behavior and therefore on its effective permeability. This is taken
into account in a semiquantitative model by Sparr and Wennerström [11].
2
LIPID SELF-ASSEMBLY
2.1
Lipid Aggregation in Water
The basic structure of the biological membrane is due to the lipids, which are
arranged in a bilayer configuration. A bilayer is a double layer of oriented
molecules (Fig. 4.). The key to the formation of such structures is the
amphiphilicity of the lipids. These amphiphilic molecules have polar
(hydrophilic) head groups and nonpolar (lipophilic) chains. Because of the dual
nature of these molecules, they self-organize in aggregates of well-defined
average shape and size. The structures of these aggregates depend on the lipid
composition, and on the physical and chemical conditions. A first and simple
way to distinguish between different phases is in terms of the chain order. In this
way one can discriminate between the liquid crystalline phases, where the
hydrocarbon chains are in a melted state (conformationally disordered), and the
solid gel and crystalline phases that have crystallized (conformationally ordered)
hydrocarbon chains.
In the majority of cases a single bilayer separates the different compartments
of living systems, but there are a number of exceptions to this rule. One clear
example is the stacked bilayers of the extracellular lipids in stratum corneum.
The majority of the lipids in stratum corneum are in the solid crystalline or gel
lamellar state [12]. Lipid molecules can be packed in different ways in the solid
state. Normal crystalline packing is a stacking of lipid bilayers, reflecting the
amphiphilicity of the molecules. The crystal structures of different ceramides
FIGURE 4 Schematic representation of some lipid bilayer structures: (a) the lamellar
(L ) liquid crystal, (b) the rippled (P ) gel, and (c) the planar (L ) gel with tilted
hydrocarbon chains.
have been studied with X-ray diffraction, showing on either V-shaped or parallel
chain packing of double-chained ceramides [13, 14]. Lipid crystals often contain
a few water molecules per lipid that are tightly bound to the lipid head group.
A gel phase can be regarded as intermediate between the crystalline and the
liquid crystalline phases. The conformationally ordered hydrocarbon chain
arrangement (which may allow for rotation of the chain) is similar to the one of
the crystalline phase, but liquid water, freely diffusing, is incorporated into the
structure, as in the liquid crystals. Owing to different packing constraints
between the lipid head groups and the crystalline chains, a number of different
gel phases with varying chain packing (i.e., different chain tilting) and varying
bilayer geometry (i.e., planar or rippled) can appear (Fig. 4b, c). The notation of
the virtually solid gel phase is perhaps a bit confusing, since the word “gel” is
more often associated with a rather soft and flexible material, like a jelly candy.
However, both the lipid gel and the jelly candy have the characteristics of a
typical gel, where one constituent has low molecular mobility (the hydrocarbon
chains of the bilayers and the cross-linked polymer in the candy) and the other
constituent moves like a liquid (the water) [15].
Lyotropic liquid crystalline phases are aggregates of amphiphilic molecules.
They can be formed when the lipids are present in a solvent, (e.g., water). The
liquid crystalline phase exhibit a long-range order, while the short-range
hydrocarbon chain packing found in the gel and crystalline phases is lost. A
liquid crystalline bilayer can be seen as a two-dimensional liquid, in which the
lipids freely diffuse in the plane of the bilayer. The interior of the bilayer also
behaves like a liquid because there is a high fraction of gauche conformation of
the acyl chains.
Lipids are known to have a very low solubility in water. Nevertheless, the
presence of water strongly affects the structure and properties of the lipid phase
(hydrophobic effect). Addition of water causes swelling of the lipid phase and
affects the curvature of the bilayers. Figure 5 shows a generalized scheme of
different liquid crystalline and micellar phases that can occur for amphiphilic
molecules (e.g., lipids, detergents, amphiphilic polymers) in aqueous solution
[16, 17]. At very low concentrations, the amphiphiles exist as monomers in

water. An increase in amphiphile concentration forces the molecules to aggregate
and to form micelles. Micelles are spherical aggregates, with the polar head
group facing the surrounding water and the hydrocarbon chain inside the
aggregate, in an oillike environment. When the concentration of the amphiphiles
is increased, the spherical shape is distorted, and the aggregates can become
elongated and rodlike. The hexagonal phases are formed by closely packed,
long, cylindrical normal or reversed micelles that are arranged in a hexagonal
pattern, resulting in a two-dimensional long-range order. Further increase in the
amphiphile concentration can result in planar bilayers. The lamellar liquid
crystalline phase consists of stacked bilayers separated by water (or any other
related solvent, e.g., glycerol). The reversed micellar and reversed hexagonal
phases exhibit the same geometry as the normal structures, although the
structures are reversed, which implies water spheres and water cylinders in a
lipid matrix. For polar lipids, the reversed structures are more common than the
normal ones.
The cubic phases are rather complex structures. The term “cubic phase” refers
to the cubic three-dimensional lattice. Several different normal and reversed
cubic structures exist. The lattice parameters of these phases are typically 10 to
20 nm [18]. The cubic phases can be either discrete or bicontinuous. The discrete
cubic phases are formed by regularly and closely packed small micelles. These
phases can occur at lipid compositions between the (reversed) micellar and the
(reversed) hexagonal phases. Unlike the discrete micellar cubic phases, the
bicontinuous cubic phases are continuous in both the lipid and the water. The
bicontinuous cubic phases are formed from rodlike or bilayer structures, and they
occur at lipid compositions between the hexagonal and the lamellar phases. The
bilayer cubic phases are characterized by a three-dimensional long-range order,
and the topology can be mathematically described by the infinite periodical
minimal surface (IPMS) [19]. The various cubic phases are highly viscous.
The bicontinuity of the bilayer cubic phases allows for diffusion in three
dimensions in both the lipid and water regions. Because of their special
structural properties, these phases have been widely explored for different
applications in, for example, drug delivery systems [20, 21] and crystallization of
membrane proteins [22, 23]. There are also numerous cubic structures in
biological membranes. Landh demonstrated more than a thousand clear
examples of cubic arrangement in cell membranes under physiological
conditions [24]. It has also been suggested that the lamellar bodies in the stratum
granulosum (Fig. 1) are cubic membranes rather than lamellar disks [25]. The
lattice parameters of the cubic structures in biological systems are about five
times larger than those observed in single lipid-water mixtures. To reach such a
high degree of water swelling, the complex biochemical system must maintain a
subtle balance between different forces causing changes in interfacial curvature,
swelling, and preservation of the cubic structure. However, it is striking that
FIGURE 5 Typical sequence of lipid morphologies obtained from lipid self-assembly in

polar solvents as a function of lipid/solvent ratio and/or temperature. (Reprinted with
permission from Ref. 17. Copyright © 1998 John Wiley & Sons Limited.)
incorporation of a simple inorganic salt such as NaSCN in the monoolein-water

cubic phase caused a doubling of the lattice parameter [26].
The sequence in which the different structures appear can be described as a

function of increasing lipid concentration or temperature (Fig. 5). The formation
of the different liquid crystalline phases is also strongly dependent on the
molecular shape of the lipids, which can be described from the effective area of
the head group, the hydrocarbon chain length, and the molecular volume [27].
The effective area per head group varies with the solvent properties (e.g.,
electrolyte concentration, pH) and the lipid concentration. This implies that the
molecular packing of the lipids varies with the lipid concentration and the
solvent properties (Fig. 5). From a geometrical point of view, the lamellar
structure of planar, stacked bilayers is the easiest one to envisage conceptually
(Fig. 4a). In this structure, the molecular geometry is cylindrical and the
molecules pack together in a planar aggregate. When the molecular geometry is
more or less conical, nonplanar aggregates are formed, such as rodlike micelles,
cubic phases, or micelles.The extracellular lipids of stratum corneum are
arranged as stacked bilayers that likely exist in both the liquid crystalline and gel
states. In the following text, focus therefore is on bilayer structures of these
types.
Lipid phase behavior can be studied with a range of complementary
experimental techniques. Lyotropic liquid crystals and gel phases can be
investigated by, for example, small-angle X-ray diffraction (SAXD), nuclear
magnetic resonance (NMR) and differential scattering spectrometry, and
calorimetric and microscope techniques [e.g., cryo-TEM (transmission electron
microscopy), freeze fracture-SEM (scanning electron microscopy), AFM (atomic
force microscopy)] [15]. Wide-angle X-ray diffraction (WAXD) is a suitable
technique for determining the crystalline structures of the solid state lipids [16].
2.2
Why Are Lipid-Water Phases Formed?
The large variety of aggregate structures signifies a range of competing forces.
Lipid aggregation in water is driven by the need to minimize unfavorable
interactions between the solvent and the lipophilic part of the lipid while the
polar lipid head groups are surrounded by water. One can expect that exposure of
the lipophilic core to the water is minimum for reversed or planar structures.
However, the lipid head groups repel each other, thus favoring normal structures
with high curvature (see Fig. 5). This effect is most dominant for large polar
head groups and ionic lipids.
To describe the mechanisms of formation of different lipid aggregates, it is
necessary to have a detailed description of the interaction forces between and
within the aggregates. This is a research field in its own, and much theoretical
and experimental work has been undertaken to elucidate the mechanisms and
their consequences [27]. The interaggregate force is a combination of several
different repulsive and attractive components, where the nature and the range of
the interaction depend on the particular properties of the system. The attractive
van der Waals (vdw) force is present between all particles and surfaces, as well as
between the hydrocarbon chains in the bilayers [28]. In a lamellar phase of
electrically neutral bilayers, like the ceramide bilayers, the repulsive interaction
is dominated by a short-range (1–3 nm), exponentially decaying force that is
generally called the hydration force. There is a debate about the molecular
mechanism behind this interaction. Some attribute the force to thermal excitations
of the lipid-water interface, suggesting that these provides an entropic source of
the repulsive interaction [29]. Others believe that the forces are the result of
structural effects due to ordering of the water molecules between the bilayer
surfaces [30, 31].
The hydration force is also present between charged bilayers. However, at
separations larger than around 1 nm it is exceeded by the more long-range,
exponentially decaying electrostatic double-layer repulsive force. When two
charged surfaces approach each other, the electrostatic double layers begin to
overlap. The counterions are then forced into a smaller space, which results in an
entropic repulsive interaction. The electrostatic double-layer interaction between
two parallel planar surfaces can be calculated from the Poisson-Boltzmann (PB)
equation [15]. In both the liquid crystalline and the gel and crystalline states, the
vdw attraction between the hydrocarbon chains is relatively weak, and the
structure is held together by the strong interactions in the polar sheet of the
bilayer. The interaction between the polar head group and the water is therefore
of uttermost importance for the formation of liquid crystalline aggregates.
There are several different techniques for measuring colloidal forces between
surfaces, and the most commonly used are the surface force apparatus [27],
atomic force microscopy [32], and the osmotic stress technique [33]. The surface
force apparatus and AFM permit direct measurements of the forces (attractive
and repulsive) between molecularly smooth surfaces or surfaces covered with
bilayers of insoluble amphiphiles. In osmotic stress measurements, the swelling
of the lipid phase is measured by X-ray diffraction at varying osmotic pressures.
The basis of this technique is to utilize the thermodynamic properties of the bulk
phase, where a desired water osmotic pressure is created by the addition of a
polymer. This technique yields a direct and accurate measurement of the force
between two bilayer surfaces. However, like all methods based on measurements
on stable bulk phases, the osmotic stress technique is feasible only when the
force is repulsive.
The amount of water taken up in the lamellar phase is mainly determined by
the interactions perpendicular to the bilayer plane, and the interaction force per
area unit, F/area, is equal to the osmotic pressure for the solvent between the two
surfaces, osm, which is physically equivalent to the chemical potential of the
water, µ w
(1)
where G is the free energy, Vw is the molar volume of the water, and nw is the
number of moles water. The water chemical potential is also related to
the relative humidity (RH) of the vapor pressure through
(2)
where R is the gas constant and Tis the temperature, and (RH is given in
percent). When a bilayer system is equilibrated with water vapor, its water
content is adjusted so that the repulsive interbilayer force corresponds to the
imposed chemical potential of the water vapor, as in Eq. (1). This way, the
swelling behavior can be directly related to the relative humidity through the
interlamellar forces [15]. It is important to note that the chemical potential of the
water is generally related to the water content in a nontrivial way. A relation
between these quantities can be obtained, for example, by isothermal
microcalorimetry [34] or the osmotic stress technique [33], or by gravimetric
techniques like the sorption microbalance [35] or the climatic chamber technique
[36].
2.3
Phase Transitions
The different lipid structures just described all occur at equilibrium under certain
conditions. The phase behavior is determined by the lipid composition, and it is
also dependent on the solution properties and the thermodynamic conditions. By
changing a variable of the system, such as temperature, water content, salt
concentration, or pH, one changes the position of the equilibrium. This could
lead to a change of the character of the present phase (e.g., the swelling of a
lamellar phase upon addition of water), or it may trigger a transition to another
phase.
Phase transformations between different lyotropic phases can be divided into
three different classes: solid-solid transitions between crystal and gel or between
different gel structures, solid-liquid transitions involving the melting and
freezing of the hydrocarbon chains, and liquid-liquid transitions between
different fluid phases.
Chain melting (the solid-liquid transition) is often referred to as the main
transition, and it is induced by, for example, changes in temperature. In the solid
state, the vdw attraction between the close-packed, solid hydrocarbon chains is
relatively weak. When the temperature is increased, the thermal motions of the
molecules overcome these attractive forces, and the hydrocarbon chains become
disordered. The temperature at which this occurs is referred to as the lipid
melting temperature. The transition temperature can be altered by changes in
FIGURE 6 Calorimetric temperature and sorption “scans” in correlation to the DMPC-

water phase diagram (a). The phase boundaries can be detected from the enthalpy changes
in the calorimetric experiments. In the DSC thermograms for DMPC-water mixtures, the
following transitions can be detected (b) gel (L ) to liquid crystal (L ) (nw/nDMPC=4) and
(c) gel (L ) to gel (P ) and gel (P ) to liquid crystal (L ) (excess water). (d) At 27°C,
transition from L to L causes a plateau of constant partial molar enthalpy of water in the
sorption enthalpy curve. (Reprinted from Ref. 39. Copyright © 2001, with permission
from Elsevier Science.)
another intensive variable such as the external pressure [37] or the osmotic
pressure [38].
Transitions between different gel phases involve a rearrangement of the
crystalline lipid molecules. This can be caused by changes in temperature or in
the hydration of the lipid head groups. The transitions between different liquid
crystalline phases takes place due to changes of the bilayer curvature. Such
transitions can be induced by changes in the water content. It may also be
thermotropically induced, since temperature increases the hydrocarbon chain
motion, which in turn promotes reversed structures.
Phase transitions can be investigated with different calorimetric techniques.
Differential scanning calorimetry (DSC) can be employed to study phase
transitions induced by variations in temperature, and isothermal calorimetric
techniques such as titration calorimetry and sorption microcalorimetry can be
used to study the effect of hydration. The combination of these techniques is a
good tool for constructing T-nw phase diagrams (Fig. 6) [39]. From the
calorimetric measurements one obtains the enthalpies for different transitions.
Representative values for the transition
TABLE 1 Representative Values for the Transition Enthalpies for the Different Phase
Transitions
Transition Htr (kJ/mol)
Lamellar cubic ca. 0.5
Lamellar reversed hexagonal 5–10
Gel liquid crystal (chain melting) 30–50
Rippled gel non rippled gel 5–10
Source: Ref 40.
enthalpies for the different phase transitions are summarized in Table 1 [40],
Generally, the liquid-liquid and solid-solid transitions involve much smaller
enthalpy effects than the chain melting. The large difference in transition
enthalpy of the transitions between different gel phases and the chain-melting
transition (gel-lamellar liquid crystal) is clearly illustrated by the DSC curve for
dimyristoylphosphatidylcholine (DMPC) in excess water (Fig. 6c). The transition
from the bicontinuous cubic phase to the lamellar structure involves a change in
bilayer curvature, while the surface continuity is preserved. The close
resemblance between the lamellar and the bicontinuous cubic phases is well
illustrated by the very small enthalpy change during the transition between these
phases (Table 1). In the cubiclamellar transitions, the occurrence of fusion
processes is minimized during the membrane formation. The occurance of
continuous foldings and unfoldings between cubic and lamellar structures has
been demonstrated in some biological membranes [24]. This has also been
suggested as a possible mechanism for the transition from the lamellar bodies of
stratum granulosum to the planar bilayers of the extracellular lipids of stratum
corneum [25].
In most experimental work, lipid phase equilibrium is studied in relation to
variations in temperature. However, for many biological applications the osmotic
pressure of water is equally relevant. The osmotic forces are important in
regulating a number of biological membrane processes. For example, membrane
fusion processes can be induced by creating a local osmotic stress [41]. The
effect of osmotic pressure on lipid phase behavior is also expected to be of
utmost importance in the phase behavior of the stratum corneum lipids.
The water content has a considerable effect on the lipid phase behavior. It was
described in connection with Figure 5 how variations in the lipid/ water ratio
affect the aggregate structures. It is also possible to trigger a transition from a
solid gel phase to a lamellar liquid crystalline phase by the addi tion of water
(Fig. 6a). The phase behavior for systems consisting of diacyl
phosphatidylcholine (PC) and water at varying water contents has been
investigated both experimentally and theoretically [42–44]. In these systems, the
addition of water can induce phase transitions between different gel phases and
between the gel and the liquid crystalline lamellar phases. Phase transitions from
gel to a lamellar liquid crystalline phase upon addition of water have also been
demonstrated for extracted stratum corneum lipids [45] and for ceramides [46].
2.4
Lipid Properties
Although the qualitative description of lipid phase behavior is based on rather
general concepts, the quantitative prediction of the lipid structure requires
information on the chemical properties of the actual lipids. There is no strict
definition of “lipid,” and there exist a large variety of different systems of
classification of different lipid classes. From the perspective of the physical
properties of the lipids, it is useful to divide lipids into groups of non polar and
polar lipids according to their surface and bulk properties [47] (Table 2). Polar
lipids are more or less amphiphilic and respond when water is added, forming
monolayers at the air-water surface. The insoluble but swelling lipids and the
soluble lipids (classes II and III of Table 2, respectively) also form
thermodynamically stable micelles or liquid crystals, as described earlier.
Stratum corneum differs compositionally from most biological membranes,
having longer chain lipids. The main classes of stratum corneum lipids are fatty
acids, ceramides, and cholesterol, but essentially no phospholipids [4, 48]. The
high fraction of solid (gel and crystalline) lipids can be explained by the very
long saturated hydrocarbon chains. The melting temperatures of lipids are
strongly influenced by the length and the saturation of the hydrocarbon chains
[47]. Long hydrocarbon chains promote the formation of a solid phase, leading to
a high melting temperature. In the fully saturated compounds, free rotation
around each carbon-carbon bond gives the hydrocarbon chain great flexibility.
These molecules can pack together tightly in crystalline arrays, allowing for
relatively strong vdw attractions between the extended hydrocarbon chains. A cis
double bond forces a kink in the hydrocarbon chain. Therefore, the unsaturated
lipids cannot pack together as tightly as the saturated lipids and, as a result, their
interactions with each other are weaker. Since less thermal energy is required to
disorder these lipids, they have a lower melting temperature than saturated lipids
of the same chain length. The more double bonds present, the lower the melting
transition. The position of the double bond also affects the melting temperature.
Double bonds that are located in the center of the molecule generally leads to a
lower melting temperature than double bonds located closer to the end of the
hydrocarbon chain [47].
Fatty acids are the simplest lipid species. Fatty acids are carboxylic acids with
hydrocarbon chains of varying length. The carboxylic acid group is polar and
accounts for the slight solubility of short-chain fatty acids in water. The phase
behavior and the solubility of fatty acids are strongly affected by variations in pH,
as the carboxylic acid groups titrate. This is further described in Section 2.5.
TABLE 2 Small’s Classification Scheme for Lipids Based on Their Surface and Bulk Properties
Source: Ref. 47
Ceramides are considered to be the most abundant lipid species in stratum

corneum, constituting approximately 40% of the total number of lipids [4]. The
ceramides are polar, uncharged lipids composed of a fatty acid linked to a
phytosphingosine backbone through an amide bond (Fig. 2c, d). At least six
different groups of ceramides (CER 1-CER 6), differing from each other by head
group architecture and fatty acid chain lengths (16C-30C) [4, 48], are present in
the stratum corneum. The ceramides found in stratum corneum are cylindrical in
shape, and therefore they most likely exclusively form lamellar phases. CER 1
(Fig. 2d) differs from the others in that it contains a linoleic acid ester linked to a
long-chain -hydroxy acid. The phase behavior of ceramide and ceramide-
cholesterol-fatty acid mixtures in water has been extensively studied by X-ray
diffraction, demonstrating two repeating distances of the crystalline lamellar
phase at approximately 6 and 13 nm [49]. It has been proposed that the presence
of CER 1, which spans more than one bilayer, is crucial for the formation of the
long periodicity, [9, 50, 51]. Basedon this observation, and on electron density
distribution calculations, the so-called sandwich model was proposed for the
arrangement of stratum corneum lipids [8]. It describes a narrow lipid layer with
fluid domains in the center of the repeating unit which is surrounded by broad
layers with crystalline structure.
Cholesterol is a major constituent in many biological membranes. It comprises
a lipid molar fraction of around 30 % in the lipid matrix of stratum corneum [4],
and in the plasma membrane of eukaryotic cells [52]. The majority of the studies
undertaken to elucidate the effects of cholesterol on lipid morphology in bulk
have been performed on systems containing various phospholipids, since these
occur frequently in biological membranes [53]. Figure 7 shows the phase diagram
of cholesterol and dipalmitoylphosphatidylcholine (DPPC) [54]. It has been
shown that the PC-cholesterol phase diagrams have a universal form, with the
main difference of a translation along the temperature axis when the acyl chain
length is varies [55]. Cholesterol is slightly miscible in gel state bilayers, and it
has been referred to as a “crystal breaker” because it disturbs the translational
order of the phospholipid molecules in the gel state [54, 56]. Cholesterol also
causes a straightening of the disordered phospholipid acyl chains in the liquid
crystal line phase and reduces the mean head group area [54]. This property is
often referred to as the stabilizing effect of cholesterol. At high cholesterol
contents, a liquid crystalline phase is formed. This phase has been denoted a
liquid ordered phase, reflecting the demonstration in several independent studies
that in this lamellar liquid crystalline phase, characterized by a relatively high
molecular lateral diffusion, there is a high degree of the acyl chain order [57].
The liquid ordered phase is stable at temperatures far below and far above the
melting temperature of the phospholipid. It has also been demonstrated that the
liquid ordered phase is stable over a large range of osmotic pressures [58]. This
is a condition highly relevant for stratum corneum lipid phase behavior because
there is a large gradient in water content—and osmotic pressure—from the blood
to the ambient air.
FIGURE 7 A T-Xchol phase diagram for DPPC-cholesterol in excess water, where Xchol is
the mole fraction of cholesterol with respect to the total number of lipid molecules. Three
different phases occur in the phase diagram: the disordered liquid crystalline phase (L ),
the ordered liquid crystalline phase (L (o)), and the ripple gel (P ) phase. (Reprinted with
permission from Ref. 54. Copyright © 1990, American Chemical Society.)
The liquid ordered phase is not specific for mixtures of saturated PCs and
cholesterol, and it has been demonstrated for mixtures of cholesterol and, for
example, unsaturated PCs [55] and fatty acids [59]. It is also believed to be
associated with important biological functions. For example, the liquid ordered
phase has been suggested to be decisive for the formation of the so-called lipid
rafts in cell membranes [60]. It was recently suggested that a cholesterol-rich
phase similar to the liquid ordered phase is present in the extracellular lipids of
stratum corneum [10]. However, there is still no experimental evidence for the
formation of a liquid ordered phase in mixtures of cholesterol and lipids with
very long hydrocarbon chains. It has been demonstrated that the miscibility of
cholesterol in lipid monolayers strongly depends on the hydrocarbon chain
length of the lipid [61, 62]. The highest miscibility has been demonstrated for
saturated acyl chains of 14 to 17 carbons [63]. The variation in miscibility can be
explained by steric packing constraints of cholesterol molecules and the
hydrocarbon chains of the lipids.
2.5
Solution Properties and Lipid Phase Behavior
The properties of the solution are also crucial for the lipid phase behavior. pH is
an important condition, and particularly when titrating molecules like the fatty
acids are considered. At low pH, the insoluble undissociated fatty acids are
present, while at higher pH the dissociated soaps are formed. This implies a
variation in the charge of the lipid head groups with pH that affects both
molecular shape and phase behavior. The balance between oleic acid and sodium
oleate is a good illustration (Fig. 8) [64, 65]. When the fraction of the oleate soap
increases, so does the interfacial charge density. The negatively charged head
groups are repelling each other, and thus exerting larger effective head group
areas (cf. Fig. 5). This promotes the formation of normal phases rather than the
reversed structures. The fatty acid:soap, a molecular compound that forms
between the fatty acid and its soap, swells in water and forms a lamellar phase.
Fatty acids in dilute monomeric solution have a pKa of 4.6 to 4.8. The pKa is
altered when the lipid concentration is changed, since the dissociation is affected
by the aggregation of the lipids. It is well known that acids attached at the lipid-
water interface are increasingly difficult to ionize the higher the negative charge
density [66]. Since titration implies an increase in surface charge density, this
effect will be more pronounced at higher pH, resulting, in turn, in an increased
apparent dissociation constant [67, 68].
The presence of electrolytes also affects the phase behavior. The addition of an
electrolyte increases the osmotic pressure of the aqueous solution. This generally
leads to a dehydration and consequently reduced swelling of the lipid phase. It
can possibly also induce phase transitions. The presence of salt also has direct
electrostatic effects through direct interactions at the polar lipid-water interface
[27]. The accumulation of chaotropic (structure-breaking) electrolytes (e.g., SCN
, I ) or urea at the lipid-water interface may, in fact, lead to an effective
swelling of the lipid phase [26, 69]. For charged lipid aggregates, the interaction
force between the bilayers is dominated by the electrostatic repulsion. The
screening effect of the salt ions causes the range of the electrostatic repulsion to
be reduced in the presence of salt, implying a reduced swelling. Furthermore, the
cationic salt ions accumulate close to the negatively charged lipid surface. This
leads to a decrease in head group interaction, which promotes smaller effective
head group areas and the formation of planar or reversed structures (cf. Fig. 5).
The addition of noncharged molecular species may also affect phase behavior.
Such effects can be important in developing an understanding of the effect of
transdermal drug delivery formulations on the lipid phase behavior of stratum
corneum. For example, the addition of nonmiscible polymers (i.e., large
polymers that do not penetrate the lipid-water phase) causes an increased
osmotic pressure of the aqueous solution [15] that can affect the swelling of the
lipids (Sec. 2.2). It is also possible to alter the phase equilibria by adding a small
hydrophobic or amphiphilic component. It has been demonstrated that the
addition of lipophilic molecules (like alkanes, alcohols, fatty acids, and charged
alkyl compounds) can alter the chain-melting transition according to its relative
solubility in the different phases. The addition of short-chain molecules that
prefer the lamellar liquid crystalline phase lowers the melting temperature, while
the addition of long-chain molecules cause it to increase [70].
FIGURE 8 Phase diagrams for oleic acid, sodium oleate, and water at 20°C. The
different phases in the diagrams are normal (L1) and reversed (L2) micelles, and cubic
(I2), reversed hexagonal (HII), lamellar (L ), normal hexagonal (HI), and gel phases [64, 65].
(Reprinted from Ref. 64. Copyright © 1995, with permission from Elsevier Science.)
2.6
Lipid Monolayers
Monolayers can be thought of as one half of a bilayer, and they can be used as
simplified and well-defined models for investigating intermolecular interactions
between lipids in membranes. When insoluble surface active lipids are spread on
a water surface, they spontaneously form a monolayer at the airwater interface.
The conventional way to characterize the lipid monolayers is to measure the
lateral surface pressure s, as a function of molecular area, A. This is
traditionally done on a Langmuir surface balance [16]. The orientation, packing,
and physical state of the lipids depend on the air-water interfacial area. A
decrease of the vapor-liquid interface induces phase transitions into different
states of the monolayer. Figure 9 shows a schematic surface pressure-area ( s-A)
isotherm. At low surface pressures, the molecules are far apart and are
considered to lie almost parallel to the surface. This state can be modeled as a
two-dimensional gas. As the monolayer is compressed, a liquid expanded phase
is induced, followed by a liquid condensed phase, until finally a solid lipid
monolayer is formed. At even higher surface pressures the monolayer will often
collapse. The chain order in the different phases is schematically illustrated in
Figure 9.
FIGURE 9 Hypothetical surface pressure-area ( s-A) isotherm including gaseous, liquid

expanded, liquid condensed, and solid phases. The chain order in the different phases is
schematically illustrated.
The liquid expanded monolayer can be considered to be a fluid phase in which

the hydrocarbon chains have a high conformational disorder, while the liquid
condensed and solid phases have small cross-sectional areas per lipid and highly
ordered hydrocarbon chains. Comparisons of the chain order and the head group
areas with those seen in bulk lamellar phases suggest that the molecular
properties of the liquid expanded, liquid condensed, and solid monolayers are
similar to those found in the liquid crystalline, gel, and crystalline bilayers,
respectively. [16] Fatty acids have served as a classical system for monolayer
studies. By recording s-A isotherms at different temperatures, one can construct
the phase diagrams for one-component fatty acid monolayers. [71, 72] Fatty acid
monolayers show very rich phase behavior with many different condensed
phases. The structural diversity of these monolayers has been thoroughly
investigated by X-ray diffraction [73].
3
DOMAIN FORMATION IN LIPID BILAYERS AND
MONOLAYERS
3.1
Phase Segregation and Domain Formation
The bilayers in biological membranes have complex compositions of different
lipid species of varying character. It is well known that multicomponent lipid
mixtures can undergo phase segregation as a result of nonideal mixing of the
lipids. Segregation can also be triggered by changes in thermodynamic
conditions such as temperature, ionic strength, or osmotic pressure. This leads to

lateral heterogeneity and the formation of domains of different chemical
composition. Differentiation into segregated lipid domains has been established
in model lipid membranes and in biological membranes [74–76]. Domain
formation has also been suggested to be decisive for the barrier properties of the
extracellular bilayers in stratum corneum [7].
Domain formation can be regarded analogous to phase segregation in bulk
mixtures. It can therefore be predicted from the equilibrium bulk phase behavior.
In a mixture containing several lipid species, domain formation can occur if the
different lipids are not totally miscible. If the chain lengths of the individual
lipids in a mixture differ by more than four carbon atoms, they tend to segregate
completely [72]. In the case of partially miscible lipids, domains of different
chemical composition are expected. A small amount of cholesterol is soluble in
PC bilayers (Fig. 7). At intermediate cholesterol content, a cholesterol-rich phase
coexists with a phospholipid-rich phase, while at high cholesterol content a
single phase is formed. It has been demonstrated that the domain formation in
phospholipid-cholesterol monolayers correlates rather well to the bulk phase
behavior [61]. Similar behavior has also been demonstrated for fatty acid-
cholesterol monolayers [62]. Domain formation can also occur in one-
component lipidbilayers [77, 78]. For example, one can expect coexisting
domains in the DMPC bilayers at compositions and temperatures corresponding
to the two-phase regions in the phase diagram in Figure 6a.
Phase diagrams can be used to predict domain formation, although they do not
give any information on the size and the shape of the domains. As described in
the domain mosaic model of the stratum corneum lipids, the transport properties
depend not only on the relative amount of the different phases, but also on the
size and shape of the domains. To study domain formation in model lipid
membranes, a range of different techniques have been used. Lateral diffusion
studies can detect domains [79, 80]. Domain formation can also be detected from
fluorescence quenching [81] and scattering studies [73]. To obtain information
on the size, shape, and lateral arrangement of the domains, visualizing
techniques, such as AFM [82], fluorescence microscopy [83], and Brewster
angle microscopy [84] can be used. Lipid monolayers at the air-water interface
or deposited mono-, bi-, or multi-layers can be used as model systems for such
investigations.
3.2
Domain Shape and Size
The size, shape, and lateral arrangement of a domain depend on its lipid
composition and can be regulated by variations of different thermodynamic
variables. Theoretically, the domain shape can under certain circumstances be
described as a balance between long-range dipole-dipole effects that cause
repulsion between the domains, and the line tension (free energy per domain
FIGURE 10 Topographic AFM image (8×8 µm2) of the transferred monolayers of (a)
palmitic acid-lignoceric acid, molar ratio 1:1, and (b) palmitic acid-lignoceric acid-
cholesterol, molar ratio 1:1:0.01. The only difference in composition between these two
samples is the presence of a small amount of cholesterol in (b). It is clearly observed that
the cholesterol has a large effect on the shape of the domains. This illustrates the line
active effect of cholesterol. (Reprinted with permission from Ref. 62. Copyright © 1999,
American Chemical Society.)
interfacial length, cf. surface tension), which favors compact circular shapes [83].
The domain shape can also reflect the molecular packing of lipids. One example
of this is the two-dimensional chiral crystals observed in mono-layers of chiral
DPPC [85]. It is however important to bear in mind that domain shape and size
are usually not equilibrium properties. For example, the irregular shaped
domains observed in Figure 10 are most likely not the equilibrium structures.
The shape of these domains can be attributed to a very slow relaxation, due to a
diffusion-limited aggregation process.
A large number of studies on lipid domain formation have shown that the size
of the domains varies with the lipid composition. To elucidate the effect of
different stratum corneum lipids on domain formation, monolayers of fatty acids,
cholesterol, and ceramides have been investigated [62, 77, 86, 87]. Figure 10a
shows an AFM image of a deposited monolayer of two fatty acids of different
chain lengths [palmitic acid, C16:0, and lignoceric acid, C24:0, molar ratio 1:1].
Owing to the large difference in hydrocarbon chain lengths, these lipids are
totally immiscible. The height difference between the domains was measured to
1.1 nm, corresponding to the difference in hydrocarbon chain length of the two
fatty acids. The shape of the fatty acid domains is strongly affected by a small
amount of cholesterol (Fig. 10) [62]. This effect can be explained by the line
active (cf. surface active) properties of cholesterol. A line active substance
reduces the line tension around two-dimensional domains, decreasing the
tendency to smooth and shorten the domain boundaries. The cholesterol
molecule consists of one rigid steroid skeleton with low conformational freedom
and one flexible (isooctyl) chain (Fig. 2b). As a consequence of its chemical
FIGURE 11 Topographic AFM images (2×2 µm2) of transferred monolayers of (a) C24
CER 3-cholesterol, molar ratio 1:1, and (b) C24 CER 3-lignoceric acid-cholesterol, molar
ratio 1:1:1. Small rectangular domains are present in the C24 CER 3-cholesterol
monolayers. When lignoceric acid (C24:0) is incorporated in the ceramide domains, these
become less regular in shape, although the size and the lateral arrangement is retained.
(Reprinted in part with permission from Ref. 87. Copyright © 2001. American Chemical
Society.)
structure, cholesterol locates preferentially at the solid-liquid domain interfaces

and reduces the line tension [88]. Line active properties have also been
demonstrated for some proteins [89] and for impurities like dyes [90].
The domains in ceramide-cholesterol monolayers are very small and regular in
shape and size [86, 87]. These domains differ significantly from the relatively
large domains observed in fatty acid- cholesterol and phospholipid-cholesterol
monolayers [63, 77]. Very small rectangular domains have been observed in
monolayers of cholesterol and synthetic ceramides related to group III of the
natural ceramides of the skin (Fig. 11a) [91]. These domains were interpreted as
two-dimensional single ceramide crystals. The short edges of the smallest
rectangular domains measured 10 nm, which corresponds to fewer than 20 lipid
molecules. Similar small-scale domains have also been observed in monolayers
composed of ceramide, cholesterol, and fatty acids (Fig. 11b) [87], and in
monolayers composed of extracted pigskin ceramides and cholesterol [86].
Together, these results indicate that for monolayers including both ceramides and
cholesterol, the tendency to form small solid domains is very high. This tendency
is quite remarkable, and may have some functional implications. It has been
suggested that the presence of small-scale domains in cell membranes plays an
important role in different biological functions. The so-called lipid raft model,
proposed in 2000 [92], describes how small solid domains (rafts) form platforms
in the membrane that support numerous cellular events in membrane traffic and
signal transduction. These domains are highly enriched in sphingolipids and
cholesterol [81]. It has also been proposed that a small amount of ceramides
significantly stabilizes the formation of such domains [93]. According to the raft
model, specific transmembrane proteins may preferentially be located in the raft

domains.
Different AFM studies on domain formation in the lipid monolayers of model
systems of stratum corneum lipids clearly show several coexisting phases [86, 87].
This is consistent with domain formation as suggested in the domain mosaic
model (Fig. 2). In evaluating these results, one should, however, be aware that a
lipid monolayer is a simplified model of the bulk phase bilayers. Such a model
neglects the interactions between the mono-layers in the bilayer, and between the
adjacent bilayers. Yet these are factors that could affect the phase behavior.
Furthermore, it was not possible to distinguish between liquid and solid phases in
the AFM studies. It is therefore appropriate to combine the monolayer studies
with results from complementary techniques, like X-ray diffraction and NMR.
Domains with a size of a few tens of square micrometers have been detected by
micro-Raman mapping in bulk mixtures of ceramides, cholesterol, and fatty
acids [94]. The presence of coexisting phases in bulk has been demonstrated in
extracted stratum corneum lipids and in model systems of the stratum corneum
lipids by a range of different techniques [12, 95, 96]. Direct comparisons of X-
ray diffraction and AFM results have shown that the lipid miscibility in
monolayers of model stratum corneum lipids is consistent with bulk phase
behavior [12, 86, 87].
4
LIPID STRUCTURE AND MEMBRANE FUNCTION
4.1
Lipid Phase Behavior and Barrier Properties
The main function of a membrane is to control the exchange of matter between
the surrounding regions. Lipid bilayers form the basic structure of biological
membranes. The lipid bilayer can be seen as a two-dimensional solvent for
hydrophobic membrane proteins. Solutes can be selectively transported across
the membranes in specific channels, and selective pumps can be created by
membrane-soluble proteins and enzymes. The lipid bilayer also provides an
efficient permeability barrier that prevents uncontrolled passive diffusion
between the surrounding regions. The efficiency of such bilayer barriers is
mainly determined from the lipid phase behavior and the domain formation in
the bilayers. The physical properties of the lipids also affect the mechanical
properties of the membrane.
Stratum corneum lipids have an arrangement of stacked bilayers parallel to the
skin surface. A stack of bilayers provides an effective barrier to passive diffusion
of polar molecules and also acts as a sink for nonpolar substances, impeding on
their transport between different membrane systems. In contrast to, for example,
the cubic phases, the stacked bilayers are not continuous in either the lipid or the
water region and thus do not provide any channels or pores to diffusing
substances.
The diffusional flux of molecules across the membrane is determined from the
membrane permeability and the concentration gradient of the diffusing
molecules. The permeability Pi of a molecular species i in a lipid bilayer is
determined from its diffusion coefficient Di, the partition coefficient between the
apolar part of the bilayer and the aqueous layers Ki and the bilayer thickness,
L:Pi,=KiDi/L. There are large variances in permeability toward different
molecular species. The diffusion coefficient of most small molecules is of the
same magnitude in the interior of a fluid (liquid crystalline) bilayer and in bulk
solvent [97, 98]. The main explanation for the variation in permeability between
different solutes in liquid crystalline bilayers is therefore not the difference in the
diffusion coefficients, but rather the differences of the partition of the diffusing
molecules into the bilayer. This implies that although the mobility of a molecule
is high in the membrane, a low average concentration will result in a low net
transport.
The transport rate of a substance across a lipid bilayer also depends on the
physical state of the lipids in the bilayer. The diffusion coefficients in a gel phase
bilayer are at least two orders of magnitudes smaller than in the liquid crystalline
phase bilayers [99, 100]. This can be explained by the low mobility of the solute
in the solid gel bilayer. Moreover, one can expect the partition coefficient
between the gel phase bilayers and water to be considerably lower than the
partition coefficient between liquid crystalline bilayers and water [101].
Together, the low molecular motion in the solid interior of the bilayer, and the
low partition into the bilayers, result in a very low permeability of the gel phase
bilayers.
The domain formation has an impact on the transport rate across membranes
consisting of stacked bilayers, and parallel pathways through the different
domains must be taken into account in considerations of the diffusional flux
across such membranes. The magnitude of each of these fluxes depends on the
fraction of each phase. The diffusion rate across the whole bilayer stack also
depends on the size of the domains in the segregated bilayers. The molecules
favor the route in which the resistance to diffusion is lowest. In bilayers of
coexisting liquid crystalline and gel domains, the molecules will mainly choose
the route through the liquid domains, and the solid gel or crystalline domains can
be regarded as obstructions (cf. Fig. 2). This leads to a longer diffusion pathway,
due to lateral diffusion in the aqueous layer parallel to the bilayer surface.
Consequently, the size and the shape of the domains affect the efficient
permeability of the membrane. If the domains are very small, the tortuosity
effect should be considered to be negligible. One can expect that the tortuosity
effect is of greater significance for the solute flux than for the water flux, as the
lateral diffusion in the aqueous layers does not significantly contribute resistance
for the water flux. The obstruction effect should be particularly important for
nonpolar molecules that have a low solubility in the aqueous layers. The
presence of segregated domains of differing permeability can potentially explain

the very low effective permeability of stratum corneum [102].
Domain formation in the bilayers leads to a lengthening of the diffusion pathway
and is thus expected to lower the effective permeability. Still, several studies
have demonstrated that segregated bilayers have higher permeability than one-
phase liquid bilayers [100, 103, 104]. This phenomenon can be attributed to an
increase in permeability at the domain boundaries. From this, one can conclude
that domain shape and size (factors that determine the interfacial boundary
lengths) have an important impact on the permeability properties of membranes.
This phenomenon is often attributed to defects or lateral density fluctuations of
the lipid molecules at the domain boundaries [105, 106]. One molecular
explanation for enhanced permeability can be the accumulation of line active (cf.
surface active) substances along the domain boundaries. This would lead to a
different composition, and possibly also a higher permeability in these regions.
The line active substance can be the diffusing solute itself and/or a bilayer
component like cholesterol (Sec. 3.2).
To elucidate the effect of domain formation on the membrane permeability,
one must consider the combined effect of the longer diffusion pathway and the
increased permeability due to the presence of domain boundaries. This is not
trivial, and a quantitative prediction requires information on the lipid
composition, size, and interfacial boundary lengths of the domains. It is also
possible that the presence of the diffusing substance affects domain size and
shape.
4.2
Responding Membranes: Interplay Between Lipid Phase
Behavior and Permeability
In a simple model of lipid membranes, the lipid bilayer is regarded as a well-
defined, uniform permeation barrier separating different compartments. This
kind of model neglects the response in lipid phase behavior to the gradient of the
diffusing substance. As discussed earlier, lipid phase behavior can be altered by
small changes in the physical or chemical environment of the lipids. Therefore,
one can expect a variation in the arrangement of the lipid molecules in the
bilayers along the gradient of the diffusing substance. In other words, the flux is
not only a function of the solute-membrane interaction (the partition coefficient)
and the state of the lipid bilayers in absence of the diffusing substance, but it is
also dependent on structural changes along the transmembrane solute gradient.
The human skin separates two very different regions, which implies large
gradients in several chemical and physical variables. It has been shown that the
lipid composition [107], salt concentration [108], and pH [109] vary between the
lower and the upper parts of the stratum corneum. The most important function of
the stratum corneum barrier is to limit water evaporation from the body. There is
a large difference between the water-rich tissue inside the body and the relatively
dry outside environment. This implies a large gradient in water content, hence in
the chemical potential of water, across the stratum corneum. Gradients of other
molecular species such as metabolic gases are also expected, as well as a
gradient in temperature. Additional gradients can be introduced by the
application of pharmaceutical or cosmetic formulations at the skin surface. Based
on these observations, one can expect not only a diffusional flux over the skin,
but also changes in bilayer properties along the gradients. The variation in lipid
structure along the gradients would also affect the effective permeability of the
skin. A quantitative description of such effects requires an understanding of the
response in lipid phase behavior to the gradients.
In a model for responding membranes, the lipid equilibrium phase behavior is
related to a gradient in the chemical potential of water across a membrane of
stacked bilayers [11, 110]. The model allows for a coupling between the steady
state flux of water and the thermodynamic response of the lipids to the local
chemical potential of water. A gradient in the chemical potential of water across
the bilayer stack implies a gradient in the interlamellar forces [Eq. (1)]. This leads
to a variation of the lipid phase behavior and aqueous layer swelling along the
bilayer stack. Figure 12 gives a schematic representation of the conditions for
this model. The upper side of the bilayer stack is exposed to air with a specified
relative humidity (RH), which can be related to the water chemical potential [Eq.
(2)]. The lower side of the stack is exposed to an aqueous environment,
represented by physiological saline solution (corresponding to RH 99.5%).
From knowledge on lipid phase behavior in homogeneous mixtures, it is possible
to predict the variation in phase behavior in the bilayer stack. A gradient in the
chemical potential of water induces a heterogeneous swelling of the stack and
may induce phase transitions between different phases in the stack (see, e.g, the
phase diagram for DMPC, Fig. 6a). The structural transformations along the
gradient result in a nonlinear transport behavior.
Figure 13 illustrates some quantitative results for water and solute flux across
a bilayer stack of binary phospholipid bilayers. When the relative humidity on
the upper side of the stack varies, the phase behavior of the lipid bilayers in the
stack will respond. At high relative humidities, all bilayers are in the liquid
crystalline state, while at low relative humidities, the lipid bilayers in the upper part
of the membrane are in the gel state. The permeability of water and solute
molecules is much lower in gel phase bilayers than in liquid crystalline phase
bilayers [99]. Consequently, the effective permeability of the membrane is
considerably reduced when the relative humidity is decreased.
A comparison between calculated water flux and experimental data for water
flux through stratum corneum [111] shows qualitatively similar responses to the
boundary condition of relative humidity (Fig. 13a). At low relative humidities
the water flux remains virtually constant even though the gradient is increased.
This mechanism prevents a massive water loss during very dry conditions. In the
theoretical model, this effect is explained by the increased fraction of gel phase
bilayers. The response of the experimental water flux in stratum corneum to the
FIGURE 12 Schematic representation of the membrane consisting of stacked bilayers, in

the presence of a gradient in chemical potential of water, µ w. On the lower side of the
stack (z=0), the chemical potential corresponds to a physiological saline solution
(corresponds to RH 99.5 %). On the upper side of the stack (z=W), the chemical potential
is determined from the relative humidity of air =RT In(RH). The lipid bilayers in the stack
respond to the local µ w. This leads to an inhomogeneous swelling of the membrane. The
gradient in µ w can also induce phase transitions (From Ref. 11.)
gradient in the chemical potential of water is qualitatively consistent with the
response of the calculated flux, suggesting that the mechanism described in the
model is also a mechanism operating for the skin lipids.
The diffusional transport of small dissolved molecules through a bilayer stack
is also affected by a gradient in the chemical potential of water (Fig. 13b). When
the upper bilayers are in the gel state, the flux is about one order of magnitude
lower than when all bilayers are in liquid crystalline state. It can be concluded
that a control of the boundary conditions in the chemical potential of water can
be used as a switch for membrane permeability. Increased permeability upon
increasing humidity at the boundary has also been demonstrated for in vivo
penetration through stratum corneum (occlusion effect) [16, 112]. This
mechanism is used in transdermal drug delivery systems.
It has been established that there is also a relatively large gradient in pH across
the stratum corneum [109]. The effect of pH on fatty acid phase behavior is
important for the understanding of stratum corneum phase behavior (Sec. 2.5).
There is a gradient in pH across stratum corneum from pH 7 in the viable
epidermis to pH 5 on the stratum corneum surface. In the innermost parts of
FIGURE 13 Calculated water and solute flux through a DLPC-DMPC (XDMPC=0.5,

T=30°C) bilayer stack as a function of relative humidity. (a) Comparison between
experimental data for water flux over stratum corneum from Blank et al. [111] (dotted
curve, right-hand axis) and the calculated water flux through a bilayer stack (solid curve,
left-hand axis). When comparing the curves it is important to note that the relative
humidity at which the curve starts to level off is determined by the properties of the
particular lipid system. The nonlinearity of the calculated curve can be attributed to the
phospholipid phase transitions from a liquid crystalline to a gel phase at decreasing water
chemical potentials. However, similar phase behavior has also been demonstrated for
extracted stratum corneum lipids [45] and for ceramides [46]. In the calculations, the flux
through the corneocytes and the crystalline lipids was neglected. This can explain why the
calculated flux is about two orders of magnitudes larger than the experimental flux. (b)
Calculated solute flux over the bilayer stack. (From Ref. 11.)
stratum corneum, a fraction of the fatty acids should be ionized, and the bilayer
surface is expected to have a relatively high charge density. Moving outward,
across the stratum corneum, the drop in pH will reduce the ionization of the fatty
acids, and thus the surface charge. The decrease in lipid head group repulsion
may also lead to decreased acyl chain fluidity of the lipids. This would lead to a
decrease in permeability [68].
Numerous studies have been reported on methods for enhancing the
permeability of the stratum corneum, persuant to the development of effective
drug delivery systems [113, 114]. For example, it has been shown that inclusion
of fatty acids in transdermal formulations enhances the permeability of a wide
range of compounds through skin. The enhancing effect is greatest for fatty acids
with low melting points, like oleic acid and lauric acid [115]. It has been
suggested that permeability may be enhanced because the fluid fatty acid is
incorporated into the stratum corneum lipid bilayer, where it induces a larger
fraction of fluid domains, thus increasing the permeability [114]. The addition of
nonlamellar forming amphiphiles could possibly also induce new structures
(cubic or hexagonal) in the lipid matrix of stratum corneum. The formation of
nonlamellar morphology would enhance permeability for both polar or nonpolar
substances [115]. In many applications of transdermal drug delivery, the dosage
is very high, and it can affect the bilayer phase behavior along the solute
concentration gradient. If the solute substance has a higher solubility in the liquid
crystalline phase that in the gel phase, a phase transition can be triggered along
the solute gradient. This offers another route to the control of membrane
permeability.
5
CONCLUSIONS
Many questions about the structure, formation, and function of the stratum
corneum remain unresolved. The function of stratum corneum is in one sense
obvious: to protect us from uncontrolled passage of substances through the skin.
The fact that stratum corneum allows for nonnegligible water transport signifies
that it is not a completely impermeable barrier. If we accept diffusional transport
over stacked bilayers as the probable mechanism for water transport, a coupled
flux of oxygen and carbon dioxide must also follow from the same arguments,
which may be essential for metabolic processes in the regions just below stratum
corneum. This line of reasoning implies a need for a construct at the molecular
level that maintains the delicate balance between protection and permeability.
The transport of both water and gases is driven by chemical potential
gradients. This is especially obvious for water, where a wet inside continuously
changes to a more or less dry outside. Such a gradient in the chemical potential
of water may not only act as a driving force for the transport of water but also
may affect the structural properties of the lipids (i.e., lead to solid-liquid
transformations and changes in swelling). These gradients can be included in a
model based on physicochemical principles of skin permeability in a relatively
straightforward way. The results from such a model of water flux give a
semiquantitative agreement with experimental results, indicating the need for a
description of stratum corneum lipid matrix more comprehensive than just its
chemical composition and its structural implications.
The formation of the lipid matrix from the lamellar bodies is another
important issue to consider. This process is accompanied by a simultaneous
transformation of lipids from phospholipids in the lamellar body to ceramides in
stratum corneum. To us it seems plausible that these chemical changes imply
changes in structure, say from liquid to solid hydrocarbon chains, which in turn
may lead to domain formation.
The complex lipid mixture with some ceramides having very long
hydrocarbon chains makes the most simple structure of stacked bilayers less
probable (although the bilayer certainly is a major building block). The high
content of cholesterol also deserves special attention, since cholesterol in lipid
mixtures is known to create dense but fluid bilayers. The “liquid-ordered”
structure formed in cholesterol-lipid mixtures may thus be an important
component of the lipid matrix that serves as a permeation route for water and
other diffusing substances.
ACKNOWLEDGMENTS
We acknowledge Nadia Merclin, Johanna Bender, and Wesley Schaal for
valuable comments on the manuscript.
REFERENCES
1. Michaels, A.S., Chandrasekaran, S.K. and Shaw, J.E. Drug permeation through
human skin: theory and in vitro experimental measurements. AICh. J. 1975; 21:
985–996.
2. Elias, P.M. Epidermal lipids, barrier function, and desquamation. J. Invest.
Dermatol. suppl. 1983; 80:44–49.
3. Engblom, J. On the phase behavior of lipids with respect to skin barrier function.
Doctorial Thesis. Lund University, 1996.
4. Wertz, P.W., Kremer, M. and Squier, C.A. Comparison of lipids from epidermal
and palatal stratum corneum. J. Invest. Dermatol. 1992; 98:375–378.
5. Nilsson, G.E. On the measurement of evaporative water loss. Methods and clinical
applications. Medical dissertation. Linköping University, 1977.
6. Hatcher, M.E. and Plachy, W.Z. Dioxygen diffusion in the stratum-corneum—an
EPR spin-label study. Biochim. Biophys. Acta 1993; 1149:73–78.
7. Forslind, B. A domain mosaic model of the skin barrier. Acta Dermatol Venereol.
1994; 74:1–6.
8. Bouwstra, J.A., Dubbelaar, F.E.R., Gooris, G.S. and Ponec, M. The lipid
organisation in the skin barrier. Acta Dermatol Venereol. 2000; suppl 208: 23–30.
9. Kuempel, D., Swartzendruber, D.C., Squier, C.A. and Wertz, P.W. In vitro
reconstitution of stratum corneum lipid lamellae. Biochim Biophys. Acta 1998;
1372:135–140.
10. Norlén, L. Skin barrier structure and function: the single gel phase model. J. Invest.
Dermatol. 2001; 117:830–836.
11. Sparr, E. and Wennerström, H. Responding phospholipid membranes— interplay
between hydration and permeability. Biophys. J. 2001; 81:1014–1028.
12. Bouwstra, J.A., Gooris, G.S., Cheng, K., Weerheim, A., Bras, W. and Ponec,
M.Phase behavior of isolated skin lipids. J. Lipid Res. 1996; 37:999–1011.
13. Dahlén, B. and Pascher, I.Molecular arrangements in sphingolipids. Crystal
structure of N-tetracosanoylphytosphingosine. Acta Crystallogr B 1972; 28:2396–
2404.
14. Dahlén, B. and Pascher, I.Molecular arrangements in sphingolipids. Thermotropic
phase behaviour of tetracosanoylphytosphingosine. Chem. Phys. Lipids 1979; 24:
119–133.
15. Evans, D.F. and Wennerström. H. The Colloidal Domain, Where Physics,
Chemistry, Biology and Technology Meet. 2nd ed. New York. VCH Publishers
1999.
16. Larsson, K. Lipids—Molecular Organization, Physical Functions and Technical
Applications. Dundee, Scotland: Oily Press 1994.
17. Jönsson, B., Lindman, B., Holmberg, K. and Kronberg, B. Surfactants and
polymers in aqueous solution. Chichester. John Wiley & Sons 1998.
18. Lindblom, G. and Rilfors, L. Cubic phases and isotropic structures formed by
membrane-lipids—Possible biological relevance. Biochim. Biophys. Acta 1989;
988:221–256.
19. Andersson, S., Hyde, S.T., Larsson, K. and Lidin, S.Minimal surfaces and
structures: from inorganic and metal crystals to cell membranes and biopolymers.
Chem. Rev. 1988; 88:221–242.
20. Engström, S.Cubic phases as drug delivery systems. Abstr. Pap. Am. Chem. Soc.
1990; 200:45-POLY.
21. Shah, J.C., Sadhale, Y. and Chilukuri, D.M. Cubic phase gels as drug delivery
systems. Adv. Drug Deliv. Rev. 2001; 47:229–250.
22. Larsson, K. and Lindblom, G.Molecular amphiphile bilayers forming a cubic phase
in amphiphile–water systems. J. Dispersion Sci. Technol. 1982; 3:61–66.
23. Landau, E.M. and Rosenbusch, J.P. Lipidic cubic phases: a novel concept for the
crystallization of membrane proteins. Proc. Natl. Acad. Sci. U.S. A. 1996; 93:
14532–14535.
24. Landh, T.Cubic cell membrane architetures. Doctoral thesis, Lund University,
1996.
25. Norlén, L.Skin barrier formation: the membrane folding model. J. Invest.
Dermatol. 2001; 117:823–829.
26. Takahashi, H., Matsuo, A. and Hatta, I. Effects of chaotropic and cosmotropic
solutes on the structure of lipid cubic phase: monoolein-water systems. Mol. Cryst.
Liquid Cryst. 2000; 347:475–482.
27. Israelachvili, J. Intermolecular and Surface Forces. 2nd ed. Academic Press 1992.
28. Parsegian, V.A., Fuller, N. and Rand, R.P. Measured work of deformation and
repulsion of lecithin bilayers. Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 2750–2754.
29. Israelachvili, J. and Wennerström, H. Entropic forces between amphiphilic surfaces
in liquids. J. Phys. Chem. 1992; 96:520–531.
30. Marcelja, S. and Radic, N. Repulsion of interfaces due to boundary water. Chem.
Phys. Lett. 1976; 42:129–130.
31. Rand, R.P. and Parsegian, V.A. Hydration forces between phospholipid bilayers.
Biochim. Biophys. Acta 1989; 988:351–376.
32. Ducker, W.A., Senden, T.J. and Pashley, R.M. Direct measurement of colloidal
forces using an atomic force microscope. Nature 1991; 353:239–241.
33. Leneveu, D.M., Rand, R.P. and Parsegian, V.A. Measurement of forces between
lecithin bilayers. Nature 1976; 259:601–603.
34. Markova, N., Sparr, E., Wadsö, L. and Wennerström, H. A calorimetric study of
phospholipid hydration. Simultaneous monitoring of enthalpy and free energy. J.
Phys. Chem. B 2000; 104:8053–8060.
35. Binder, H., Kohlstrunk, B. and Heerklotz, H.H. A humidity titration calorimetry
technique to study the thermodynamics of hydration. Chem. Phys. Lett. 1999; 304:
329–335.
36. Jendrasiak, G.L. and Hasty, J.H. The hydration of phospholipids. Biochim.
Biophys. Acta 1974; 337:79–91.
37. Estep, T.N., Mountcastle, D.B., Biltonen, R.L. and Thompson, T.E. Studies on the
anomalous thermotropic behaviour of aqueous dispersions of
dipalmitoylphosphatidylcholine-cholesterol mixtures. Biochemistry 1978; 17:
1984–1989.
38. Smith, G.S., S.E.B., Safinya, C.R., Plano, R.J. and Clark, N.A. X-ray structural
studies of freely suspended ordered hydrated DMPC multimembrane films. J.
Chem. Phys. 1990; 92:4519–4529.
39. Markova, N., Sparr, E. and Wadsö, L. On application of an isothermal sorption
microcalorimeter. Thermochim. Acta 2001; 374:93–104.
40. Cevc, G. Phospholipids Handbook. New York. Marcel Dekker, 1993.
41. Hui, S.W., Kuhl, T.L., Guo, Y.Q. and Israelachvili, J. Use of poly(ethylene glycol)
to control cell aggregation and fusion. Colloid Surfaces B 1999; 14: 213–222.
42. Janiak, M.J., Small, D.M. and Shipley, G.G. Temperature and compositional
dependence of the structure of hydrated dimyristoyl lecithin. J. Biol. Chem. 1979;
254:6068–6078.
43. Guldbrand, L., Jönsson, B. and Wennerström, H. Hydration forces and phase
equilibria in the dipalmitoyl phosphatidylcholine-water system. J. Colloid Interface
Sci. 1982; 89:532–541.
44. Ulmius, J., Wennerström, H., Lindblom, B. and Arvidson, G. Deuteron nuclear
magnetic resonance studies of phase equilibria in a lecithin-water system.
Biochemistry 1977; 16:5742–5745.
45. Gay, C.L., Guy, R.H., Golden, G.M., Mak, V.H.W. and Francoeur, M.L.
Characterization of low-temperature (ie, <65°C) lipid transitions in human stratum-
corneum. J. Invest. Dermatol. 1994; 103:233–239.
46. Shah, J., Atienza, J.M., Rawlings, A.V. and Shipley, G.G. Physical properties of
ceramides—effect of fatty-acid hydroxylation. J. Lipid Res. 1995; 36:1945–1955.
47. Small, D.M. The Physical Chemistry of Lipids. New York: Plenum Press, 1986.
48. Norlén, L., Nicander, I., Lundsjö, A., Cronholm, T. and Forslind, B.A new HPLC-
based method for the quantitative analysis of inner stratum corneum lipids with
special reference to the free fatty acid fraction. Arch. Dermatol. Res. 1998; 290:
508–516.
49. Bouwstra, J.A., Gooris, G.S., Bras, W. and Downing, D.T. Lipid organization in
pig stratum corneum. J Lipid Res 1995; 36:685–695.
50. Bouwstra, J.A., Gooris, G.S., Dubbelaar, F.E. R., Weerheim, A.M., Ijzerman, A.P.
and Ponec, M. Role of ceramide 1 in the molecular organization of the stratum
corneum lipids. J. Lipid Res. 1998; 39:186–196.
51. Mérigoux, C. Étude de l’organisation supramoléculaire de la peau par diffusion de
rayons X et microscope électronique. Doctoral thesis,l’Université Paris-Sud XI
1997.
52. Yeagle, P.L. Cholesterol and the cell membrane. Biochim. Biophys. Acta 1985;
882:267–287.
53. Bloom, M., Evans, E. and Mouritsen, O.G. Physical properties of the fluid lipid-
bilayer component of cell membranes: a perspective. Rev. Biophysics 1991; 24:
293–397.
54. Vist, M.R. and Davis, J.H. Phase equilibria of cholesterol/
dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic resonance and
differential scanning calorimetry. Biochemistry 1990; 29:451–464.
55. Thewalt, J.L. and Bloom, M. Phosphatidylcholine-cholesterol phase-diagrams.
Biophys. J. 1992; 63:1176–1181.
56. Ipsen, J.H., Karlström, G., Mouritsen, O.G., Wennerström, H. and Zuckermann,
M.J. Phase equilibria in the phosphatidylcholine-cholesterol system. Biochim.
Biophys. Acta 1987; 905:162–172.
57. Lindblom, G., Johansson, L.B. and Arvidsson, G. Effect of cholesterol in

membranes. Pulsed nuclear magnetic resonance measurements of lipid lateral
diffusion. Biochemistry 1981; 1920:2204–2207.
58. Sparr, E., Hallin, L., Markova, N. and Wennerstöm, H. Phospholipid-cholesterol
bilayers under osmotic stress. Biophys. J. 2002; 83:2015–2025.
59. Pare, C., and Lafleur, M. Formation of liquid ordered lamellar phases in the
palmitic acid/cholesterol system. Langmuir 2001; 17:5587–5594.
60. Brown, D.A. and London, E. Structure and function of sphingolipid- and
cholesterol-rich membrane rafts. J. Biol. Chem. 2000; 275:17221–17224.
61. Hagen, J.P. and McConnell, H.M. Liquid-liquid immiscibility in lipid monolayers.
Biochim. Biophys. Acta 1997; 1329:7–11.
62. Sparr, E., Ekelund, K., Engblom, J., Engström, S. and Wennerström, H. An AFM
study of lipid monolayers: II. The effect of cholesterol on fatty acids. Langmuir
1999; 15:6950–6955.
63. Slotte, J.P. Lateral domain heterogeneity in cholesterol/phosphatidylcholine
ylcholine monolayers as a function of cholesterol concentration and
phosphatidylcholine acyl chain length. Biochim. Biophys. Acta 1995; 1238:118–
126.
64. Engblom, J., Engström, S. and Fontell, K. The effect of skin penetration enhancer
Azone on fatty acid-sodium soap-water mixtures. J. Controlled Release 1995; 33:
299–305.
65. Seddon, J.M., Bartle, E.A. and Mingins, J. Inverse cubic liquid-crystalline phases
of phospholipids and related lyotropic systems. J.Phys.-Condensed Matter 1990;
2:SA285-SA290.
66. Tatulian, S.A. lonization and ion binding. In: Cevc, G. ed. Phospholipids
Handbook. New York: Marcel Dekker, 511–552 1993.
67. Engblom, J., Engstrom, S. and Jonsson, B. Phase coexistence in cholesterol fatty acid
mixtures and the effect of the penetration enhancer Azone. J. Controlled Release
1998; 52:271–280.
68. Lieckfeldt, R., Villalain, J., Gomez-Fernandez, J.-C. and Lee, G. Apparent pKa of
the fatty acids within ordered mixtures of model human stratum corneum lipids.
Pharm. Res. 1995; 12(11):1614–1617.
69. Sanderson, P.W., Lis, L.J., Quinn, P.J. and Williams, W.P. The Hofmeister effect
in relation to membrane lipid phase stability. Biochim. Biophys. Acta 1991; 1067:
43–50.
70. Lohner, K. Effects of small organic molecules on phospholipid phase-transitions.
Chem. Phys. Lipids 1991; 57:341–362.
71. Ställberg-Stenhagen, S. and Stenhagen, E. Phase transitions in condensed
monolayers of normal chain carboxylic acids. Nature 1945; 3956:239–240.
72. Bibo, A.M. and Peterson, I.R. Phase diagrams of monolayers of the long chain fatty
acids. Adv. Mater. 1990; 2:309–311.
73. Kenn, R.M., Böhm, C., Bibo, A.M., Peterson, I.R., Möhwald, H., Als-Nielsen, J.
and Kjaer, K. Mesophases and crystalline phases in fatty acid monolayers. J.Phys.
Chem. 1991; 95:2095–2097.
74. Bergelson, L.O., Gawrisch, K., Feretti, J.A. and Blumenthal, R. Domain
organisation in biological membranes. Mol. Membr. Biol. 1995; 12:1–162.
75. Rietveld, A. and Simons, K. The differential miscibility of lipids as the basis for the
formation of functional membrane rafts. Biochim. Biophys. Acta 1998; 1376:467–
479.
76. Mouritsen, O.G. and Jorgensen, K. Small-scale lipid-membrane structure:
simulation versus experiment. Curr. Opin Struct. Biol. 1997; 7:518–527.
77. Ekelund, K., Sparr, E., Engblom, J., Wennerström, H. and Engström, S. An AFM
study of lipid monolayers: I. Pressure-induced phase behavior of single and mixed
fatty acids. Langmuir 1999; 15:6946–6949.
78. McConnell, H.M.,Tamm, L.K. and Weis, R.M. Periodic structures in lipid
monolayer phase transition. Proc. Natl. Acad. Sci. USA 1984; 81:3249–3253.
79. Almeida, P.F.F., Vaz, W.L. C. and Thompson, T.E. Lateral diffusion and
percolation in 2-phase, 2-component lipid bilayers—topology of the solid-phase
domains inplane and across the lipid bilayer. Biochemistry 1992; 31: 7198–7210.
80. Bloom, M. and Thewalt, J.L. Time and distance scales of membrane domain
organization. Biochemistry 1995; 33:6707–6715.
81. Xu, X.L. and London, E. The effect of sterol structure on membrane lipid domains
reveals how cholesterol can induce lipid domain formation. Biochemistry 2000; 39:
843–849.
82. Dufrene, Y.F. and Lee, G.U. Advances in the characterization of supported lipid
films with the atomic force microscope. Biochim. Biophys. Acta 2000; 1509:14–41.
83. McConnell, H.M. Structures and transitions in lipid monolayers at the air-water
interface. Annu. Rev. Phys. Chem. 1991; 42:171–195.
84. Wolthaus, L., Schaper, A. and Möbius, D. Microcrystallinity of solid-state Langmuir-
Blodgett films of saturated fatty acids studied by scanning force microscopy and
Brewster angle microscopy. J.Phys. Chem 1994; 98: 10809–10813.
85. Weis, R.M. and McConnell, H.M. Two-dimensional chiral crystals of phospholipid.
Nature 1984; 310:47–49.
86. Ten Grotenhuis, E., Demel, R.A., Ponec, M., Boer, D.R.,Van Miltenburg, J.C. and
Bouwstra, J.A. Phase behavior of stratum corneum lipids in mixed Langmuir-
Blodgett monolayers. Biophys. J. 1996; 71:1489–1399.
87. Sparr, E., Eriksson, L., Bouwstra, J.A. and Ekelund, K. AFM study of lipid
monolayers: III. Phase behavior of ceramides, cholesterol and fatty acids.
Langmuir 2001; 17:164–172.
88. Keller, D.J., McConnell, H.M. and Moy, V.T. Theory of superstructures in lipid
monolayer phase transitions. J. Phys. Chem. 1986; 90:2311–2315.
89. Haas, H. and Möhwald, H. Specific and unspecific binding of concanavalin A at
monolayer surfaces. Thin Solid Films 1989; 180:101–110.
90. Miller, A. and Möhwald, H. Diffusion limited growth of crystalline domains in
phospholipid monolayers. J Chem Phys 1987; 86:4258–4265.
91. Ekelund, K., Eriksson, L. and Sparr, E. Rectangular solid domains in ceramide-
cholesterol monolayers—2D crystals. Biochim. Biophys. Acta. 2000; 1464:1–6.
92. Simons, K. and Ikonen, E. Functional rafts in cell membranes. Nature 1997; 387:
569–572.
93. Xu, X.L., Bittman, R., Duportail, G., Heissler, D., Vilcheze, C. and London, E.
Effect of the structure of natural sterols and sphingolipids on the formation of
ordered sphingolipid/sterol domains (rafts). J.Biol. Chem. 2001; 276:33540–33546.
94. Percot, A. and Lafleur, M. Direct observation of domains in model stratum corneum
lipid mixtures by Raman microspectroscopy. Biophys. J. 2001; 81:2144–2153.
95. Fenske, D.B., Thewalt, J.L., Bloom, M. and Kitson, N. Models of stratum corneum
intercellular membranes—2H NMR of macroscopically oriented multilayers.
Biophys. J. 1994; 67:1562–1573.
96. Moore, D.J., Rerek, M.E. and Mendeisohn, R. Lipid domains and orthorhombic
phases in model stratum corneum: evidence from Fourier transform infrared
spectroscopy studies. Biochem. Biophys. Res. Commun. 1997; 231:797–801.
97. Marrink, S.J. and Berendsen, H.J. C. Permeation process of small molecules across
lipid membranes studied by molecular dynamics simulations. J.Phys. Chem. 1996;
100:16729–16738.
98. Angelico, R., Balinov, B., Ceglie, A., Olsson, U., Palazzo, G. and Soderman, O.
Water diffusion in polymer-like reverse micelles. 2. Composition dependence.
Langmuir 1999; 15:1679–1684.
99. Lawaczeck, R. On the permeability of water molecules across vesicular bilayers.
J.Membr. Biol. 1979; 51:229–261.
100. Xiang, T.X. and Anderson, B.D. Phase structures of binary lipid bilayers as
revealed by permeability of small molecules. Biochim. Biophys. Acta 1998; 1370:
64–76.
101. Mesquita, R., Melo, E., Thompson, T.E. and Vaz, W.L. C. Partitioning of
amphiphiles between coexisting ordered and disordered phases in two-phase lipid
bilayer membranes. Biophys. J. 2000; 78:3019–3025.
102. Forslind, B., Engström, S., Engblom, J. and Norlén, L. A novel approach to the
understanding of human skin barrier function. J. Dermatol. Sci. 1997; 14:115–125.
103. Cruzeiro-Hansson, L. and Mouritsen, O.G. Passive ion transport of liquid
membranes modelled via lipid-domain interfacial area. Biochim. Biophys. Acta
1988; 944:63–72.
104. Clerc, S.G. and Thompson, T.E. Permeability of dimyristoyl phosphatidylcholine/
dipalmitoyl phosphatidylcholine bilayer—membranes with coexisting gel and
liquid-crystalline phases. Biophys. J. 1995; 68:2333–2341.
105. Georgallas, A., Macarthur, J.D., Ma, X.P., Nguyen, C.V., Palmer, G.R., Singer,
M.A. and Tse, M.Y. The diffusion of small ions through phospholipid-bilayers.
J.Chem. Phys. 1987; 86:7218–7226.
106. Van Hoogevest, P., De Gier, J. and De Kruiff, B. Determination of the size of the
packing defects in dimyristoylphosphatidylcholine bilayers, present at the phase
transition temperature. FEBS Lett. 1984; 171:160–164.
107. Bonté, F., Saunois, A., Pinguet, P. and Meybeck, A. Existence of a lipid gradient in
the upper stratum corneum and its possible biological significance. Arch.
Dermatol. Res. 1997; 289:78–82.
108. Forslind, B., Lindberg, M., Roomans, G.M. and Pallon, J. Aspects on the
physiology of human skin: studies using particle probe analysis. Micros. Res.
Tech. 1997; 38:373–386.
109. Öhman, H. and Vahlquist, A. In vivo studies concerning a pH gradient in human
stratum corneum and upper epidermis. Acta Dermatol. Venereol. 1994; 74:375–
379.
110. Sparr, E. and Wennerström, H. Diffusion through a responding lamellar liquid
crystal. A model of molecular transport across stratum corneum. Colloids Surf.
2000; 19:103–116.
111. Blank, I.H., Moloney, J., Emslie, A.G., Simon, I. and Apt, C. The diffusion of water
across the stratum corneum as a function of its water content. J. Invest. Dermatol.
1984; 82:188–194.
112. Roberts, M.S. and Walker, M.Water: the most natural penetration enhancer. In
Walters, K.A.and Hadgraft, J., ed. Pharmaceutical Skin Penetration Enhancement.
New York. Marcel Dekker. 1993.
113. Johnson, M.E., Mitragotri, S., Patel, A., Blankschtein, D. and Langer, R.
Synergistic effects of chemical enhancers and therapeutic ultrasound on transdermal
drug delivery. J.Pharm. Sci. 1996; 85:670–679.
114. Potts, R.O., Mak, V.H., Guy, R.H. and Francoeur, M.L. Strategies to enhance
permeability via stratum corneum lipid pathways. Adv. Lipid Res. 1991; 24:175–
212.
115. Francoeur, M.L., Golden, G.M. and Potts, R.O. Oleic-acid—its effects on stratum-
corneum in relation to (trans) dermal drug delivery. Pharm. Res. 1990; 7:621–627.
116. Engblom, J. Bicontinuous cubic phase: A model for investigating the effects on a
lipid bilayer due to a foreign substance. Chem. Phys. Lipids 1996; 84: 155–164.
4
Stratum Corneum Lipid Structure: Insights
from NMR and FTIR Spectroscopic Studies
Ya-Wei Hsueh and Jenifer Thewalt
Simon Fraser University, Vancouver, British Columbia, Canada
Neil Kitson
University of British Columbia, Vancouver, British Columbia,
Canada
David J.Moore
Unilever Research US, Edgewater, New Jersey, U.S.A.
The lipid matrix of the stratum corneum (SC) provides several critical
physiological functions, the most important of which is protecting the body from
dehydration. In recent years our knowledge of lipid composition in the stratum
corneum, and of the enzymatic pathways by which these lipids are generated, has
increased significantly. It is now well established, for example, that the SC lipid
matrix consists primarily of saturated free fatty acids, cholesterol, and arange of
ceramide species but does not appear to contain any phospholipids or significant
amounts of unsaturated lipids [1]. As discussed elsewhere, many elegant X-ray
and electron microscopy studies have yielded a picture of the stratum corneum
lipid matrix in which these extracellular lipid components can be seen to consist
of stacked lamellae, within which are embedded the corneocytes of the SC. It is
obvious that full comprehension of SC barrier physiology necessitates a detailed,
understanding of lateral and transverse lipid organization at the molecular level.
However, a detailed description of molecular organization, and the specific
structural and functional role of lipid heterogeneity in the SC, remain to be
experimentally determined.
While molecular spectroscopy techniques are routinely employed in cell
membrane biophysics for probing details of molecular arrangement and specific
molecular interactions, these techniques have not been widely applied to the field
of SC biophysics, and specifically molecular organization in the SC lipid barrier.
Of particular note is the paucity of fundamental studies utilizing well-
characterized model systems that can provide detailed understanding of lipid
interactions and behavior. Such studies are complementary to other
measurements utilizing physiological samples that are intact but which do not, by
their fundamental nature, provide specific molecular information.
Our laboratories are exploring the molecular biophysics of the stratum
corneum by utilizing nuclear magnetic resonance (NMR) spectroscopy (JT, NK,)
and Fourier transform infrared (FTIR) spectroscopy (DJM) methods to probe the
58 HSUEH ET AL.
nature of lipid organization in a range of biomimetic systems. These studies are

designed to probe specific aspects of lipid organization in the stratum corneum
lipid barrier. The chapter provides a general overview of some of this work and
the type of information that can be gleaned from NMR and FTIR spectroscopic
studies of stratum corneum model membranes. This includes a description of the
basic complementary information that can be garnered from biophysical NMR
and FTIR spectroscopic studies of lipid membranes and a brief discussion of
current findings.
1
STRATUM CORNEUM BARRIER LIPIDS
Before describing our biophysical techniques, it is necessary to briefly introduce
the major lipids of the SC corneum barrier and emphasize how different their
properties are from much more widely studied phospholipid membranes. The
major SC lipid classes are saturated free fatty acids, cholesterol, and two classes
of ceramides, the sphingosines and the phytosphingosines. There exist at least
eight distinct ceramide species, depending on the degree of head group
hydroxylation; the precise details of lipid composition in the barrier are described
elsewhere in this book and are not covered here. The biophysical studies
described in this chapter utilize model systems consisting of non-hydroxy fatty
acid sphingosine ceramide (often referred to as human ceramide 2 or bovine
ceramide III), palmitic acid (C16:0), and cholesterol: the structures of these
molecules are shown in Figure 1. The details of preparing the stratum corneum
model lipid mixtures are described in the primary literature, although it is worth
noting that our studies are routinely conducted in systems at pH 5.2 to 5.5 (skin
barrier pH) and are equimolar mixtures. It is important to emphasize the
relatively low level of hydration in these systems, by which we mean that only a
small amount of water seems to bind or hydrate the lipid head groups. The
swelling of bilayers routinely observed with phospholipid membranes does not
occur with skin barrier lipids. These are considerably more hydrophobic than
phospholipids of equivalent chain length, which is entirely consistent with their
role in providing a water permeability barrier for the body.
STRATUM CORNEUM LIPID STRUCTURE 59
FIGURE 1 Chemical structures of the SC model membrane components.
2
2H NMR: GENERATION AND INTERPRETATION OF
SPECTRA OBTAINED FROM SC MODEL
MEMBRANES
2.1
NMR Signal: Physical basis
To perform an NMR experiment requires an NMR coil, a static magnetic field
H0 and an alternating magnetic field that is generated by applying an alternating
current of frequency v to the NMR coil. The sample is placed inside the NMR
60 HSUEH ET AL.
FIGURE 2 Left: the Zeeman energy levels of a spin l=1 nucleus in a magnetic field. To
excite transitions among these levels the sample should be irradiated at the “Larmor”
frequency, vL. Right: an exaggerated depiction of the modification of the Zeeman energy
levels by the quadrupolar interaction. Since the transition from m=1 to m=0 now has a
different E than the transition from m=0 to m= 1, the quadrupolar spectrum is a doublet.
(Orientation effects are not included in this diagram).
coil within the static magnetic field H0. Each nuclear spin in the sample interacts
with H0, producing the so-called Zeeman levels (or energies), Em= n H0m (for
2H nuclear spin I=1, m=1, 0, 1, see left-hand side of Fig. 2).
At equilibrium, there are more spins favoring the orientation parallel to H0

(m=1 state) than antiparallel to H0 (m= 1 state), thus the net magnetization lies
in the H0 direction. This equilibrium state is perturbed by an alternating magnetic
field with frequency v, which is applied to the sample for a certain duration—
typically a few microseconds. If v is selected so that hv=energy difference
between adjacent levels (i.e., v= nH0/2 =vL), then transitions between the
Zeeman levels can be induced. During this process, the magnetization is tipped
down from the H0 direction to a plane perpendicular to H0, and once the
alternating field has been shut off, the magnetization precesses in this plane.
Since this precession occurs within the NMR coil, it generates a measurable
voltage, the NMR signal. The frequency with which this voltage oscillates
determines the spectrum.
2.2
2H NMR Spectrum: Quadrupole Couplings
The interaction dominating the 2H NMR spectrum is the quadrupolar interaction,
which originates from the electrostatic interaction between nuclear charge and
the electric field associated with charges from other atoms. The 2H nucleus
possesses a quadrupole moment, owing to the nonspherically symmetric
distribution of its nuclear charge. This nuclear quadrupole moment, Q, interacts
with the electric field gradient at the 2H nucleus, giving rise to the quadrupolar
interaction. The electric field gradient (EFG) is defined by the chemical structure
of the 2H-labeled molecule, and for chain-labeled lipids the C—D bond
determines it. For 2H the dipolar and chemical shift interactions are much
smaller than the quadrupolar interaction and thus will be neglected.
In a large static magnetic field, the quadrupolar interaction can be treated as a

first-order perturbation on the Zeeman interaction. For an axially symmetric EFG
such as that associated with a C—D bond, the energy levels are Em= n H0m+
(e2qQ/4)(3m2–2)(3cos2 1)/2, where is the angle between the EFG principal
axis (i.e., C—D bond vector) and H0[2]. Thus the Zeeman levels are shifted by
the quadrupolar interaction, as indicated on the right-hand side of Fig. 2b and the
spectrum becomes a doublet. In a pulverized crystalline sample, where no
molecular motion occurs, all angles will be represented, each with its own
probability. This results in a powder spectrum, the so-called Pake doublet, as
seen in Fig, 2 (for reviews, see Refs. [2] and [3]):
2.3
Line Shape for Randomly Oriented Liquid Crystalline
Microdomains
We now consider a sample consisting of a large number of liquid crystalline
microdomains whose lipid bilayer normals are randomly oriented with respect to
H0. Since axial rotation of individual lipids in liquid crystalline lamellae is very
rapid, the quadrupolar interactions are partially averaged by the motion. In this
case, the orientation of a C—D bond vector can be described by two angles: n,
the angle between the lipid bilayer normal and H0, and CD: the angle between C
—D bond vector and the lipid bilayer normal. Then Em can be rewritten as
follows:
Therefore, the energy differences between adjacent levels are
where “+” corresponds to m=0 1 transition, “–” corresponds to m=1 0

transition, and angle bracket < > , denote a time average. In the liquid crystalline
phase CD varies rapidly because of trans-gauche isomerization of the 2H-labeled
lipid chain. For a given n, we obtain a doublet— two 2H NMR resonance lines
centered about vL. Since the lipid bilayer normal is randomly distributed, the 2H
NMR spectrum will consist of a super-position of doublets representing all
possible orientations (i.e., all n). Again, the spectrum is a Pake doublet, but this
time the overall width of the spectrum would be reduced compared with the
spectrum shown in Figure 3. The two sharp peaks in the Pake doublet correspond
to i=90°, (i.e., the orientation of the membrane normal lying perpendicular to
H0). The frequency spacing between these two peaks is the quadrupolar splitting
62 HSUEH ET AL.
vQ=(3e2qQ/4) SCD, where SCD=<3 cos2 CD 1>/2, measuring the time-averaged

orientation of the C—D bond, is defined as the orientational order parameter.
2.4
2H NMR as a Probe of Membrane Phase States
Membrane lipids in aqueous environments display a variety of phases, such as
liquid crystalline, solid, gel—all associated with lamellar (typically bilayer)
membranes with different amounts of chain mobility. Nonlamellar phases are
also possible, although the biological relevance of such arrangements is
controversial. We have used 2H NMR experiments on model membranes in
which the lipid molecules have been deuterated at one or more acyl chain
positions to probe the phase of membranes. The 2H NMR spectrum of each phase
state displays distinct features. These features are associated with the lipid
chains’ molecular motions and the molecular packing arrangements that
characterize these phases. In the experiments described here 4–8, the deuterated
lipid is a 16-carbon saturated fatty acid, palmitic acid, labeled from C2 through
C16. This lipid is termed PA-d31. In the solid phase, the lipid is essentially
motionless. Lateral diffusion is negligible. The lipid chains, which are
presumably in all-trans conformation (except the end-chain methyl group, which
continues to reorient), pack closely. For example, the lipid chains of solid
dipalmitoylphosphatidylcholine (DPPC) pack in an orthorhombic crystalline
array [9]. As shown in Figure 4a, at room temperature the 2H NMR spectrum of
the SC model membrane, 1:1:1 ceramide/cholesterol/PA-d31, consists mainly of
a Pake doublet with a quadrupolar splitting of 126 kHz, typical for the solid
phase. Note that the narrower Pake doublet “riding” on the main doublet is due to
the still-mobile CD3.
In the liquid crystalline bilayer phase, lipid molecules diffuse freely in each
layer with a lateral diffusion constant of ~10–12 m2 /sec. Individual lipids within
such membranes undergo considerable motions, including axial rotation about
the lipid long axis, trans-gauche isomerizations in the hydrocarbon chain, and
nodding and shaking of the head group[10]. Owing to fast molecular
reorientations, the maximum obtainable quadrupolar splitting in the liquid phase
spectrum (Fig. 4b) is only half that observed in the solid phase. The spectrum
consists of several superimposed Pake doublets with different quadrupolar
splittings from deuterons at different segments of the PA-d31. Generally,
splittings are largest for segments near the aqueous interface (bilayer surface)
and become smaller for segments near the hydrophobic core of the membrane.
The chain conformation can be determined by using SCD calculated from the
quadrupolar splittings.
An example of a spectrum observed for nonbilayer lipids is shown in
Figure 4c. The quadrupolar splittings have completely collapsed, leaving only a
single sharp peak at the center. This type of spectrum indicates that the motions
FIGURE 3 The Pake doublet spectrum that results from a “powder” of molecules
containing C—2H bonds that are oriented randomly with respect to the magnetic field;
C—2H bonds oriented parallel or antiparallel to the field contribute to the spectrum’s
shoulders at ± 126 kHz. In their bonds oriented most probable orientation, perpendicular
to the field (i.e., C—2H bonds contribute), to the sharp peaks at ± 63 kHz).
of the labeled lipids are completely isotropic: they are tumbling rapidly and
randomly. The probable arrangement of the PA-d31 in this case is “oily droplets”
of melted fatty acid, although other arrangements of lipid in water, such as the
exotic “bicontinuous cubic” (or “plumber’s nightmare”) phase, also yield such
spectra.
The first moment, M1, also known as the average spectral width, is defined as
where v is the frequency shift from the central frequency and I(v) is the spectral
intensity First moments calculated from the spectrum of chain-labeled lipids are
exquisitely sensitive to phase changes. A significant change of M1 is usually
64 HSUEH ET AL.
FIGURE 4 The 2H NMR spectrum as a function of temperature of the model SC

membrane composed of hydrated ceramide: cholesterol: palmitic acid (in equimolar
proportion) at pH 5.2. The palmitic acid (PA) is labeled with 2H at all positions along its
chain. (a) At room temperature the spectrum indicates that the PA is essentially a solid,
barring rotation of the end methyl C[2H3]. The signal near the center of the spectrum
indicates that a small proportion of the PA is experiencing some molecular motion. (b)
When the membrane is heated, the PA undergoes a phase transition to a state
characterized by rapid molecular motion about the long axis of the molecule. This type of
anisotropic motion is characteristic of liquid crystalline membranes—the common state of
biological membranes—and results in the spectrum shown. This is a super-position of
Pake doublets having different widths, reflecting the increasing conformational motion of
the fatty acyl chain toward the membrane core. (c) At higher temperatures still, the
spectrum is a single line (see Fig. 2), indicating that the quadrupolar interactions have
been averaged to zero by the molecular motion. This isotropic motion is characteristic of
membranes transformed to small oily droplets, micelles, or cubic phases. (The latter
possibility has been ruled out by small angle X-ray diffraction).
FIGURE 5 The average 2H NMR spectral width (M1) as a function of temperature for the
SC model membrane whose spectra are shown in Figure 4. Transitions from one state to
another occur at temperatures where the spectrum narrows sharply. Two transitions, from
42 to 46°C (solid to liquid crystalline) and from 50 to 54°C (liquid crystalline to
isotropic), are evident. (Note that the conversion of the spectrum to the narrow isotropic
line is not complete until about 70°C, but the rate of conversion is slower above 54°C.)
The spectra within the transition regions are superpositions of spectra of the types shown
in Figure 4: they can be analyzed to determine the proportion of the labeled PA in each
phase present in the membrane.
observed as membranes transform from one phase to another. In Figure 5, M1

decreases significantly at two regions (40–44°C and 50– 53 °C), where the PA-
d31 component in SC model membranes undergoes transitions from the solid
phase to the liquid crystalline phase, and from the liquid crystalline phase to the
isotropic phase, respectively.
The 2H NMR technique provides quantitative knowledge of the physical state
of the labeled lipid. It has been used most widely to study membranes in the
liquid crystalline phase, where detailed information about chain conformations
can be obtained. In the solid phase, the absence of chain motion is revealed in the
observation of the largest possible quadrupolar splitting (known as the static
quadrupolar coupling constant). However, no other information is obtained for this
phase. Since 2H NMR is a “local” technique, it does not report directly on the
66 HSUEH ET AL.
identity of neighboring molecules in the model membrane. Hence in the three-

component SC model membrane, we cannot glean information about the
arrangement of the three lipids in the solid phase: we can say only that the PA-
d31 chains are essentially motionless. Similarly, in the isotropic phase, we can
say only that the PA-d31 molecules are rapidly reorienting in all directions. If the
membrane is partway through a phase change, the spectrum of the labeled lipid
can be quantitatively analyzed to determine the proportion of lipid in each phase.
This is one of the major advantages of 2H NMR: the lipid cannot hide, all spins
are seen.
Employing model membranes in which alternate components are labeled
enhances the power of 2H NMR. For SC model membranes, labeling the
ceramide component is fairly straightforward, and one can imagine that great
insights into the phase behavior of the membrane as a whole would result from a
detailed study of this alternate label. A more efficient approach, however,
employs the complementary technique of FTIR spectroscopy. Both PA-d31 and
ceramide can be studied at the same time, in the same model membrane, by
exploiting the differences in vibrational frequencies between deuterated and
protonated chains. Additionally, FTIR spectroscopy can be used to gain some
understanding of the degree to which individual lipids are segregated within the
solid phase.
3
FTIR SPECTRA FROM STRATUM CORNEUM MODEL
MEMBRANES
It is beyond the scope of this chapter to provide either a detailed description of
the components and architecture of a modern FTIR spectrometer, or the
underlying reasons enabling such instruments to provide high-quality infrared
spectra (even of traditionally infrared-unfriendly samples as aqueous
dispersions). For such information the reader is referred to a recent review
chapter [11] or to any standard text on FTIR spectroscopy. It should be
appreciated, however, that a modern FTIR spectrometer, when equipped with a
high-sensitivity photoconductive detector such as a mercury cadmium telluride
(MCT) detector cooled by liquid nitrogen, can generate high quality spectra in a
matter of seconds, from samples as small as 10 µL. Furthermore, in addition to
standard transmission sampling techniques, methods such as attenuated total
reflectance (ATR) FTIR spectroscopy and infrared reflection-absorption
spectroscopy (IRRAS) allow a variety of experimental conditions to be varied in
situ as spectra are being acquired. All such approaches have been exploited to
examine model SC membrane samples [12–14].
The infrared spectra of lipid membranes contain several features arising from
various hydrocarbon methylene (CH2) vibrations. Among these are several
modes that are extremely sensitive to the inter-and intramolecular organization
of lipid chains [15]. Furthermore, in membranes containing ceramides and fatty
acids, such as SC models, a variety of peaks can be observed arising from lipid
head group vibrations [13, 16]. These can provide insight into head group
hydrogen bonding, water binding, and fatty acid ionization. As discussed earlier,
a particularly useful aspect of the FTIR experiment is the ability to monitor
ceramide and fatty acid components simultaneously in the spectrum of an SC
model membrane. This is accomplished by preparing samples containing
protonated ceramide and deuterated palmitic acid (PA-d31), the identical sample
to that used for the NMR investigations. In the infrared spectrum of such a
sample the vibrational modes of each component are distinct and therefore can
be monitored simultaneously. A representative spectrum is shown in Figure 6. To
illustrate the utility of FTIR spectroscopic studies of SC model membranes, and
the inherently complementary nature of these studies with the NMR
investigations, the remainder of this section describes our results from studies of
a ceramide 2-PA-d31-cholesterol model membrane.
Methylene stretching vibrations, whether from CH2 or CD2 groups, are
sensitive to intramolecular conformational order (i.e., trans /gauche isomerization
of the lipid chains). This is illustrated schematically in Figure 7, which plots the
increase in methylene stretching frequency that occurs with a temperature-
induced transition from gel to liquid crystalline bilayers for a sphingomyelin
membrane. In the case of stratum corneum model membranes, these modes are
monitored for both the ceramide and PA-d31 chains, providing simultaneous and
direct measures of the conformational behavior of each component in the
membrane. The data for the SC model membrane clearly indicate that PA-d31
undergoes conformational disordering between ~40 and 55 °C, whereas the onset
of disorder (chain fluidization) for ceramide 2 does not begin until ~55°C
(Fig. 8). These data clearly suggest there are distinct lateral domains of each lipid
species within the SC model membrane [17].
The presence of a deuterated fatty acid within these model membranes can
provide insight into the intermolecular organization of both the palmitic acid and
ceramide components of these membranes. This is accomplished by exploiting a
coupling that can occur between either the rocking or bending vibrations of
methylene groups (CH2 and CD2) on adjacent lipid chains. As illustrated in
Figure 9, the lateral packing of like chains within the x-y plane of a crystalline
bilayer (i.e., orthorhombic packing) produces a doublet in the IR peaks from
bending and rocking modes. This behavior provides a direct measure of chain
mixing in these model membranes. In the case of an equimolar ideally mixed
membrane, there is no coupling between adjacent protonated and deuterated
chains, and therefore only a single peak is observed in the spectrum (for both CH2
and CD2 modes). Interestingly, however, the current SC model membrane
displays a doublet in both the fatty acid CD2 bending peak and the ceramide 2
CH2 rocking peak. The temperature dependence of this behavior is mapped out in
Figure 10. Two significant conclusions can be drawn from these studies. The
observation of splitting in CH2 and CD2 modes is direct evidence of separate
highly enriched domains of palmitic acid and ceramide 2 in the physiological
68 HSUEH ET AL.
FIGURE 6 Infrared spectrum of SC model membrane containing ceramide, PA-d31, and

cholesterol. The major methylene peaks arising from protonated and deuterated chains are
indicated.
temperature range. Both the ceramide and fatty acid domains consist of
orthorhombically packed (i.e., highly crystalline) chains.
The information inherent in the FTIR spectra of a simple SC model system
clearly provides some intriguing insights into the inter-and intramolecular
organizational possibilities in the stratum corneum lipid bilayers. An ongoing
FTIR spectroscopy study of alternative composition model stratum corneum
membrane models suggests significantly different behavior for systems
composed of other ceramide species, such as the phytosphingosines.
The FTIR experiment clearly provides significant insight into the ordered
phases of both the ceramide and fatty acid components in SC models. However,
it sheds little light on the more disordered (liquid crystalline) phase, which may
be present at low levels and yet is likely to be critical to the overall properties of
the SC lipid barrier. Direct observation of the fraction of labeled lipid present in
a more disordered phase is provided by the NMR measurement. In concert, these
techniques can provide a comprehensive description of lipid dynamics and
organization in SC models.
FIGURE 7 When a bilayer membrane undergoes a gel-liquid crystalline transition, a

characteristic increase in the methylene stretching frequency is observed in the infrared
spectrum. By monitoring this parameter, it is possible to determine the melting
temperature Tm of the transition.
4
RELEVANCE OF MODEL MEMBRANES TO THE
STUDY OF SC STRUCTURE
As mentioned in the chapter introduction, biophysical studies on membrane
structure and dynamics usually rely on simplified membranes of well-defined
composition. These are known as “model” membranes. The reasons for relying
on model membranes are both practical and philosophical: practical because the
results of experiments using model membranes can be interpreted cleanly and
without ambiguity, and philosophical in the sense that biophysicists generally
strive to understand the essentials of their system before working on the more
complex “natural” membrane.
A great deal of experimental data exists from studies of one-or two-
component lipid model membranes. Information about lipid phase “preferences”
(i.e., thermodynamics), lipid membrane structure and dynamics (e.g., lateral,
rotational, and translational diffusion within the bilayer, as well as chain
conformational motions), and lipid-lipid interactions could not be obtained
without model membranes. For example, one of the major roles of cholesterol in
mammalian plasma membranes, the creation of a thicker bilayer that is less
subject to passive leakage, was inferred from studies of model phospholipid-
cholesterol membranes. Currently much interest among membrane biologists and
biochemists concerns a particular liquid crystalline membrane phase called the
70 HSUEH ET AL.
FIGURE 8 The thermotropic behavior of the methylene stretching frequency of PA-d31

(CD2) and ceramide (CH2) in a SC model membrane (monitored simultaneously). Note
that the two components undergo their transitions at significantly different temperatures.
Chain fluidization begins at ~40 °C for PA-d31, yet does not commence until ~55 °C for
ceramide.
FIGURE 9 The lateral packing of isotopically alike lipid chains in an orthorhombic

subcell arrangement produces a doublet in both the methylene rocking and bending modes
in the infrared spectrum. The frequency of each component can be plotted to determine
the temperature at which the crystalline orthorhombic packing structure collapses. Note
that increased rotational movement along the lipid chains may cause this collapse to occur
well before the onset of chain fluidization.
“liquid ordered” phase, which is purported to be involved in the regulation of

cell signaling in natural membranes. Liquid ordered phases were first observed in
two-component phospholipid-cholesterol model membranes.
FIGURE 10 The thermotropic behavior of the methylene bending mode of PA-d31 (CD2)
and rocking mode of ceramide (CH2) in a SC model membrane (monitored
simultaneously). The presence of two peaks for each component is direct evidence of
separate crystalline domains of each lipid. Collapse of crystalline orthorhombic packing
occurs at 40 and 52 °C for the PA-d31 and ceramide components, respectively.
Although some techniques applied to model membranes are bulk techniques,

such as differential scanning calorimetry (DSC), which measure the properties of
the membrane as a whole, many rely on some form of chemical “labeling” of
lipids. Such labeling enables the researcher to look selectively at the particular
labeled entity and results in detailed molecular-level insights into structure and
dynamics. It has often been observed, for instance, that in a hydrated membrane
composed of two or more lipid components, liquid and solid membrane phases
can coexist. Labeling the individual lipids allows the determination of the amount
of each lipid in each phase. Labels can be fluorescent lipids for optical studies,
lipids with stable free radicals attached for examination by means of electron
spin resonance, or isotopically labeled lipids such as those used in the IR and
NMR experiments reviewed here. The substitution of deuterons for protons in a
lipid is a gentle form of labeling that results in only very small perturbations of
membrane structure and phase behavior.
Dangers inherent in the model membrane approach stem from the
philosophical elements of biophysicists’ motivation. If the model membrane is
poorly constructed, or if the researcher is simply unlucky, clean, unambiguous
results may be obtained that do nothing to capture the essentials of the natural
system. Fortunately in the case of SC model membranes, this hazard has been
avoided. The intercellular stacked “barrier” lamellae are a typical natural
membranes in that they contain few proteins. Studies of natural SC (see, e.g.,
Ref. 18) have revealed that the layers have an unusually crystalline
organizational motif, which contrasts dramatically with the “liquid crystalline”
state that is characteristic of almost all cell membranes. The crystalline nature of
72 HSUEH ET AL.
the lipids was also observed in the model SC membranes we studied by means of
IR and NMR. While the diversity of ceramides and fatty acids in SC is much
larger than in the SC model three-component membranes, the model SC
membrane captures at least some of the essential physical qualities of the natural
system.
REFERENCES
1. Schaefer H, RedelmeierTE. Skin Barrier: Principles of Percutaneous Absorption,
Basel: Karger, 1996.
2. Seelig J. Deuterium magnetic resonance: theory and application to lipid
membranes. Q Rev Biophys. 1977; 10:353–384.
3. Davis JH. The description of membrane lipid conformation, order and dynamics by
2H NMR. Biochim. Biophys Acta 1983; 737:117–171.
4. Thewalt J, Kitson N, Araujo C, MacKay A, Bloom M. Models of stratum corneum

intercellular membranes: the sphingolipid headgroup is a determinant of phase
behavior in mixed lipid dispersions. Biochem. Biophys Res Commun 1992; 188:
1247–1252.
5. Kitson N, Thewalt J, Lafleur M, Bloom M.A model membrane approach to the
epidermal permeability barrier. Biochemistry 1994; 33:6707–6715.
6. Fenske DB, Thewalt JL, Bloom M, Kitson N. Models of stratum corneum
intercellular membranes: 2H NMR of macroscopically oriented multilayers.
Biophys J 1994; 67:1562–1573.
7. Bouwstra JA, Thewalt JL, Gooris GS, Kitson CN. A model membrane approach to
the epidermal permeability barrier: an x-ray diffraction study. Biochemistry 1997;
36:7717–7725.
8. Abraham W, Kitson N, Bloom M, Thewalt J. Investigation of membrane structure
and dynamics by deuterium NMR: application to the stratum corneum. In: Potts
RO, Guy RH eds. Mechanisms of Transdermal Drug Delivery, New York, Marcel
Dekker: 1997; 36:163–198.
9. Ruocco MJ, Shipley GG. Characterization of the sub-transition of hydrated
dipalmitoylphosphatidylcholine bilayers: x-ray diffraction study. Biochim.
Biophys. Acta 1982; 684:59–67.
10. Gennis RB. Biomembranes. New York: Springer-Verlag, 1989.
11. Mendelsohn R, Moore DJ. IR Determination of conformational order and phase
behavior in ceramides and stratum corneum models. Methods Enzymol 2000; 312:
228–247.
12. Velkova V, Lafleur M. Influence of the lipid composition on the organization of
skin lipid model mixtures: an infrared spectroscopy investigation. Chem Phys
Lipids 2002; 1–2:63–74.
13. Rerek ME., Chen H-C, Markovic B, Van Wyck D, Garidel P, Mendelsohn R,
Moore DJ. Phytosphingosine and sphingosine ceramide headgroup hydrogen
bonding: structural insights through thermotropic hydrogen/deuterium exchange. J
Phys Chem 2001; 105:9355–9363.
14. Flach CR, Mendelsohn R, Rerek ME, Moore DJ. Biophysical studies of model
stratum corneum lipid monolayers by infrared reflection-absorption spectroscopy
and Brewster angle microscopy. J Phys Chem 2000; 104:2159– 2165.
15. Mendelsohn R, Moore DJ. Vibrational spectroscopic studies of lipid domains in

biomembranes and model systems. Chem Phys Lipids 1998; 96:141–157.
16. Moore DJ, Rerek ME, Mendelsohn R. FTIR studies of the conformational order
and phase behavior of ceramides. J Phys Chem 1997; 101:8933–8940.
17. Moore DJ, Rerek ME, Mendelsohn R. Lipid domains and orthorhombic phases in
model stratum corneum: evidence from Fourier transform infrared spectroscopy
studies. Biochem Biophys Res Commun 1997; 231:797–801.
18. Pilgram GSK, Engelsma-van Pelt AM, Bouwstra JA, Koerten HK. Electron
diffraction provides new information on human stratum corneum lipid organization
studied in relation to depth and temperature. J Invest Dermatol 1999; 113:403–409.
5
“Confidence Intervals” for the “True” Lipid
Composition of the Human Skin Barrier?
Philip Wertz
University of lowa, lowa City, lowa, U.S.A.
Lars Norlén
University of Geneva, Geneva, Switzerland
The outcome of experiments using mixtures of synthetic or extracted skin lipids
as models of skin barrier structural organization ultimately depends on the
chosen lipid composition. For experiments of these kinds, access to accurate and
precise (within a few molar percent) compositional data is therefore a
prerequisite. In the absence of such accurate and precise compositional data (cf.
Tables 1–5 at the end of the chapter), it is of fundamental importance for
biophysical model building and experimentation in general to gain a perception
of the reliability of the available data: that is, to get an idea of the “confidence
interval” within which the “true” lipid composition in the population can be
found. Such a “confidence interval” is needed to be able to judge whether a
particular experiment is worth performing. The construction of a “confidence
interval” for human stratum corneum lipid compositions thus may not only save
a lot of experimental time, and money, by avoiding meaningless experimentation,
but also may help to identify individual lipid fractions that are less well
characterized quantitatively today, toward which, consequently, more
quantitative analysis efforts may be directed. This chapter aims at identifying
such “confidence intervals” for each lipid fraction of the human skin barrier and
at pointing out difficulties involved in quantitative analytical experimentation
(e.g., contamination) that must be addressed if future experiments are to decrease
the width of these “confidence intervals.”
1
STRATUM CORNEUM LIPIDS AND THE BARRIER OF
THE SKIN
As epidermal keratinocytes move off the basement membrane and undergo
differentiation, they begin to synthesize lipids [1]. In the traditional view, much
of the lipid that accumulates with differentiation is packaged into small
organelles called lamellar granules [2]. The lamellar granules arise from the
region of the Golgi apparatus and are round to ovoid. They consist of a unit-
bounding membrane surrounding one or sometimes several stacks of internal
lamellae as well as a battery of hydrolytic enzymes. In the late stages of the
differentiation process the lamellar granules migrate to the apical end of the cell.
76 WERTZ AND NORLÉN
The bounding membrane of the lamellar granule then fuses with the cell plasma
membrane, and the contents are extruded into the intercellular space at the
boundary between the granular layer and the stratum corneum [2, 3]. The lipids
associated with the lamellar granules are largely phospholipids,
glucosylceramides, and cholesterol, with only low proportions of free fatty acids
and cholesterol esters [4–6]. After extrusion, the hydrolytic enzymes act on the
phospholipids and glycolipids to produce a mixture of ceramides, fatty acids, and
cholesterol. The free fatty acids in this barrier lipid mixture are highly saturated,
and most are more than 20 carbon atoms in chain length [7, 8].
Monounsaturated fatty acids may be transferred from phosphoglycerides to
cholesterol to produce unsaturated cholesterol esters by a lecithin: cholesterol:
acyltransferase-like enzyme. Linoleic acid may be recycled in the viable portion
of the epidermis [9].The mixture of ceramide, cholesterol, and fatty acids that
passes into the intercellular spaces of the stratum corneum rearranges as it
undergoes enzymatic processing to form tri laminar structures 13 nm wide, with
a characteristic broad-narrow-broad pattern of lucent bands, as visualized in
transmission electron micrographs prepared by using ruthenium tetroxide
fixation [10].
In addition to the lipids derived from the extruded lamellar granule contents, -
hydroxy ceramides, -hydroxy acids, and normal fatty acids are derived from the
lipids of the bounding membrane of the lamellar granule and become covalently
attached to the outer surface of the cornified envelope. It has been proposed that
within the intercellular lamellae of the stratum corneum there exist islands of gel
phase domains surrounded by a continuous liquid crystalline phase [11]. In this
“domain mosaic” model, the gel phase domains are important for limiting the
permeability of the stratum corneum, while the liquid (disordered) crystalline
phase is needed for pliability. However, the Landmann model for skin barrier
formation [3] and the domain mosaic model for skin barrier structure and
function [11] have recently been challenged by the membrane folding model
[12] and the single gel phase model [13, 14], respectively.
2
ANALYTICAL TECHNIQUES
2.1
Thin-Layer Chromatography (TLC)
Thin-layer chromatography has been the most widely used method for the analysis
of stratum corneum lipids [15–17]. In principle any solid can be used as the
stationary phase, and differential partitioning between the stationary phase and
the mobile liquid phase is the basis for lipid separation during development of
the chromatogram. In practice, silica gel has virtually always been the stationary
phase. In conventional thin-layer chromatography a glass plate is generally
“CONFIDENCE INTERVALS” FOR LIPID COMPOSITION? 77
coated with a 250 µm thick layer of silica gel G, which contains calcium sulfate
as a binder. The silica gel G is applied as an aqueous slurry, after which the
plates are baked in a 110 °C oven. The plates are then washed to push organic
contaminants to the upper edge of the plate. This is done by placing the plates in
a closed glass tank containing a polar solvent, such as chloroform-methanol (2:
1), to depth of about l cm; the solvent is allowed to wick to the top of the plate.
Plates are then air-dried before they are returned to a 110°C oven for actiyation.
Activated plates are stored in closed cabinet of storage tank over desiccant to
avoid rehydrating the activated silica gel. It is advisable to divide the adsorbant
into narrow (5–6 mm) lanes before use [18]. Dividing the silica into lanes allows
30 or more samples to be analyzed on a single 20×20 cm2 plate. The practice also
prevents radial diffusion, which allows smaller samples and improves the
accuracy of subsequent quantitation.
By means of calibrated glass capillary tubes or a Hamilton syringe, samples
are applied about 2 cm from the bottom edge of the plate. Four or 5 µL of lipid
solution is sufficient to apply lipid across a 6 mm lane. Chromatograms are
developed in closed rectangular tanks, lined with filter paper and containing
mobile phase solvent to a depth of about 1 cm. The development tanks are
prepared in advance and allowed to equilibrate before use. The filter paper liners
assist in equilibration of the liquid and vapor phases. A number of different
development regimens, generally involving multiple development steps with
different solvent systems, have been used. Usually, two different plates are
developed to quantitate all the stratum corneum lipids—one for the nonpolar
components, including cholesterol, fatty acids, and cholesterol esters, and one for
the ceramides and cholesterol sulfate. After a plate has been developed, it is air-
dried.
There are a number of development options available, but the two most widely
used involve charring induced by heating after spraying with either sulfuric acid
[18] or a cupric acetate-phosphoric acid reagent [19]. These reagents are similar
in performance. The cupric acetate-phosphoric acid reagent gives a slightly
higher carbon yield with highly saturated lipids but does cause some
overoxidation of unsaturated lipids, an effect that is not seen with sulfuric acid.
Quantitation of the chromatograms can be achieved either by means of a
scanning densitometer or by generating a digitized image of the plate and using
an image analysis system to generate carbon density profiles [18, 20]. In either
case, peak areas are related to lipid masses. However, since this is inherently not
a linear relationship, it is important to include standards on the plate so that
standard curves can be established. Since, moreover, different lipid classes give
different carbon yields, at least one standard for each lipid type needs to be
included. For example, the standard curve for cholesterol will differ somewhat
from that for ceramides, but one can use only one ceramide standard curve because
all the different structural types of ceramide give essentially the same carbon
yield.
A variation on conventional thin-layer chromatography is high-performance

thin-layer chromatography, or HPTLC [15, 17]. In HPTLC the sizes of
individual particles of silica gel are smaller and the size distribution more
uniform than in the silica gel used in conventional thin-layer chromatography.
Generally, investigators use 10×10 or 10 × 20 cm3 HPTLC plates that are
commercially available from several sources. The plates are not prepared in the
laboratory because the silica gel particles are too small to be cleared from the
lungs, and thus the dust would present a risk for silicosis. For this reason, it is
also inadvisable to use HPTLC plates for preparative purposes. The smaller and
more uniform silica particles in HPTLC do provide for a greater number of
theoretical plates per unit length of plate, and this improves the resolution.
Because the development time is considerably shorter than with conventional
thin-layer chromatography, radial diffusion is not a problem, in HPTLC, and the
adsorbant is not scored into lanes. Although samples can be applied manually
with glass capillaries, the best results are obtained when samples are applied as
streaks by means of a robotic sample applicator. Otherwise, preparation of the
plates for use, development, detection, and quantitation is essentially the same as
for conventional thin-layer chromatography. With HPTLC, all the stratum
corneum lipids can generally be quantitated from one plate [21]. For example,
CER EOH is resolved from CER NP, but CER AS and CER NH are generally not
resolved from one another (for an explanation of this ceramide nomenclature, see
[22] or the discussion in Sec. 4 of this chapter).
A relatively little used variant on thin-layer chromatography is the latroscan
[24–26]. With this instrument silica rods are used, and detection is accomplished
with a flame ionization detector. The advantages and disadvantages of the
latroscan have been reviewed [27].
2.2
High Performance Liquid Chromatography-Light
Scattering Detection (HPLC-LSD)
In HPLC, a mobile phase is forced at high pressures through a stationary phase in
a narrow column. The substances that are to be separated will partition between
the two phases (the mobile and the stationary phase) according to their
individual partition coefficients, with resulting differences in retention time in
the column. If there were no diffusion, each molecular compound would migrate
as a sharp front line. However, the compounds emerge from the column as
Gaussian-shaped peaks (in the ideal case). Diffusion parallel to the flow will
cause a symmetrical band broadening, especially for molecules with long
residence times in the column or when very low flow rates are used. Radial
diffusion causes band broadening at the column walls because the flow is lower
here than in the center of the column. Other factors that may contribute to band
broadening are irregularities of the packing material and thickness of the stationary
phase. The column efficiency is usually given as the peak width at a given
retention time: the wider the peak, the lower the column efficiency [28]. In the
normal phase mode, the stationary phase is of high polarity while the mobile
phase is more non polar.
The light-scattering detector (LSD) evaporates the solvent emerging from the
column in a stream of nitrogen or air inside a heated chamber. The solute is not
evaporated but nebulized into micrometer-sized drops that are passed through a
light beam. The amount of scattered light is proportional to the amount of
material that has been eluded. A drawback is the limited range of solvents that
can be used, since these agents must be able to evaporate in the heating chamber.
Also, the detector response is usually sigmoid and not linear and can change
slightly over time. Upon reaching optimal conditions (e.g., gas inlet pressure,
temperature of the evaporation chamber, sample size), however, the linear range
is usually fairly broad and does not present any difficulties for most applications
except that daily calibration curves must be run for each component under study
[28].
2.3
Gas Liquid Chromatography-Flame lonization Detection-
Mass Spectrometry (GLC-FID-MS)
Gas liquid chromatography (GLC) is a form of partition chromatography in
which the mobile phase is a gas and the stationary phase is a liquid. When a
sample is injected into the gas phase, it is volatilized and passed into a column
that holds the stationary liquid phase. The retention times of different
compounds in the column depend on the compounds’ relative affinities for the
stationary phase. Ideally, they emerge from the column with a Gaussian
distribution. Increased resolution is usually achieved by increasing the column
temperature during an analysis. One major drawback is that only volatile
compounds can be analyzed [29, 30].
Wall-coated open tubular (WCOT) columns usually consist of a narrow fused
silica tube (0.1–0.3 mm i.d., 25–50 m long), the inner wall of which is coated by
the liquid phase. The separative capacity of these columns is very high, but sample
size and injection technique are critical. Band broadening is a minor problem
with WCOTcolumns, since there is essentially only one flow path and the liquid
phase is more uniform in thickness than for packed columns [29, 30].
The flame ionization detector (FID) has high sensitivity, high stability, and fast
response time. In addition, it is linear over a very broad range and can be used
for most organic compounds. A current, resulting from ions that are generated by
combustion of the eluted organic compounds in a flame of hydrogen and air, is
measured as a potential difference between two electrodes and the result
transmitted to a recorder. The main variables that can exert an influence on
detector response are the flow rates of carrier gas and hydrogen and air of the
flame [29]. Since the response is related to the oxidation number of the atoms,
FID results may not be linearly related to molecular mass for all organic
substances. To compensate for this during quantitative work, researchers

calculate theoretical correction factors, based on the relative weight percent of
active carbons in the molecule [31].
Mass spectrometry (MS) is a highly valuable tool for the identification of lipids
separated by gas chromatography. In principle, organic molecules in the gaseous
phase are bombarded with electrons to form positively charged ions. These ions
will become fragmented in different ways into smaller ionized entities and
subsequently separated in a magnetic field according to their mass-to-charge
ratio. The fragmentation is not random, but takes place according to complex
rules that have been defined empirically. It is thus possible to deduce the
structure of an unknown organic compound from the specific distribution of
different fragments in the mass spectrum [29, 30]. Electrospray ionization (ESI)
was introduced in the late 1980s by Fenn. The revolutionary feature of
electrospray is that multiply charged ions are formed. The sensitivity of ESI-MS
is truly remarkable and allows for the unambiguous identification of organic
molecules (e.g., peptides and lipids) at the 10 18 mol level.
3
CONTAMINANTS IN SKIN LIPID ANALYSIS
EXPERIMENTS
Many published accounts of the composition of lipids from human stratum
corneum have been complicated by the presence of sebaceous lipids as well as
exogenous contaminants [7, 32]. When stratum corneum samples are obtained
from excised skin, there is almost always massive contamination with
triglycerides from subcutaneous fat. In addition, fatty acids are derived from the
subcutaneous triglycerides through the action of lipases on the skin surface. The
human skin surface is also generally coated with sebaceous lipids [33]. This is a
major source of squalene, wax esters, and triglycerides, and again the
triglycerides undergo hydrolysis to yield fatty acids. The sebaceous fatty acids,
mostly 16 and 18 carbons in length, contain high proportions of monounsaturated
species and variable proportions of branched chains. This is in contrast to the
stratum corneum fatty acids, which are mostly straight-chain, saturated
compounds, longer than 20 carbons.
The omnipresence of medium-chain free fatty acid contamination is
exemplified by the extreme experimental and interindividual variation in the
human forearm stratum corneum medium-chain free fatty acid fraction (<20C) in
comparisonto the long-chain free fattyacid fraction ( 20C) [8]. The medium-
chain free fatty acid fraction was dominated by C16:0, C16:l, C18:0, C18:l and
was invariably present in the blank of each of the 22 subjects analyzed (while no
long-chain free fatty acids were present in any of the 22 blanks). The extremely
large interindividual variation and the almost complete absence (< 1 mol %) in 3
out of the 22 subjects of unsaturated mediumchain free fatty acids speaks
strongly in favor of the notion that this fraction mainly is of extra endogenous
origin [8]. Reports claiming the presence in the lower stratum corneum of
significant amounts of unsaturated mediumchain free fatty acids may therefore
be viewed with some reservation.
Human sebum also contains smaller proportions of cholesterol and cholesterol
esters. In addition, precautions must be taken to avoid contamination with
environmental contaminants such as alkanes and cosmetic components. To avoid
these complications, much work has been done with the pigskin model. Young
pigs, if properly tended, can be kept clean, and their sebaceous glands are not
active. By direct heat separation of epidermis from intact skin on the carcass, it is
possible to avoid subcutaneous fat. In terms of general structure, composition,
and permeability barrier function, pig stratum corneum appears to provide a good
model for the human tissue.
Alternatively, one can use the contents of human epidermal cysts [7]. This
material represents exfoliated stratum corneum cells free of sebaceous and
environmental contamination. If carefully expressed from the capsule, the contents
can be obtained without contamination, and the lipids can be extracted and
analyzed. If cyst material is to be used, it is important to obtain the pathologist’s
report to confirm the absence of microorganisms and that the cyst was
epidermal, not trichilemmal.
4
CHEMICAL STRUCTURES OF STRATUM CORNEUM
LIPIDS
The structures of porcine stratum corneum ceramides were determined in detail
before those from human specimens [34]. With the pig ceramides, six
chromatographically distinct fractions were isolated and analyzed by a
combination of chemical, chromatographic, and spectroscopic methods. The pig
ceramides were referred to as ceramides 1 through 6, with ceramide 1 being the
least polar fraction. Each fraction contained only one structural type of ceramide,
although there was considerable heterogeneity in the lengths of the aliphatic
chains within most fractions. The least polar of the porcine ceramides, ceramide
fraction 1, consisted of -hydroxy-acids containing 30 to 34 carbons, amide
linked to a mixture of sphingosines and dihydrosphingosines. This ceramide also
has a fatty acid ester linked to the -hydroxyl group, a high proportion of which
was linoleic acid. This unusual ceramide has been referred to an acylceramide,
and it is thought to play an essential role in determining the ultrastructural
organization of the intercellular lipid of the stratum corneum [35–38]. The other
porcine ceramides contained combinations of sphingosines or phytosphingosines
with aminelinked normal or -hydroxy acids. Pig ceramides 4 and 5 both
consisted of -hydroxy acids amide-linked to sphingosines. They differed in that
ceramide 4 contained mainly -hydroxy acids with 24 to 28 carbons, while
ceramide 5 contained a-hydroxypalmitic acid almost exclusively.
When the human epidermal ceramides were fractionated and analyzed by the
same methods used for the porcine ceramide, a similar, but not identical,
chromatographic profile was obtained, and all same ceramides were found [39].
Human ceramide fraction 3 gave a somewhat broader band on thin-layer
chromatography than its porcine analogue, and there was often a shoulder at the
leading edge of this band. The human ceramide fraction with mobility similar to
pig ceramides 4 and 5 was broad and did not resolve into two separate bands.
This fraction was referred to as ceramide 4/5. Finally, the human material with
mobility similar to pig ceramide 6 split into two incompletely resolved bands,
which were labeled ceramides 6I and 6II. More recently it has been shown that
the human ceramides include several struc tural types that contain a
phytosphingosine analogue, 6-hydroxysphingosine [41, 41]. Thus, human
fraction 3 contains an acylceramide with 6-hydroxy-sphingosine as the base
component. Similarly, ceramide 4/5 contains, in addition to the -hydroxy acid-
sphingosine conjugates, a ceramide composed of normal fatty acids amide-linked
to 6-hydroxysphingosine, and ceramide 6II consists of -hydroxy acids amide-
linked to 6-hydroxy-sphingosines.
Both the original pig ceramide nomenclature system and the original human
ceramide nomenclature, as well as slight modifications thereof, are still in use in
the literature. This can create a great deal of confusion. A solution to this problem
was proposed in 1994 by Motta et al. [22]. In the proposed nomenclature system,
the amide-linked fatty acid is designated as N, A, or O to indicate normal, -
hydroxy-, or -hydroxy acid, respectively. The base component is designated S,
P, or H to designate sphingosine, phytosphingosine, or 6-hydroxysphingosine,
respectively. It is understood that sphingosines are generally accompanied by
dihydrosphingosines in the ceramides. When an ester-linked fatty acid is present,
this is designated with a prefix E. Thus pig ceramide 1, the acylceramide,
becomes CER EOS. Ceramide 2, with normal fatty acids amide-linked to
sphingosines, becomes CER NS, and so forth.
After ceramides, cholesterol and free fatty acids are the other major lipids in
epidermal stratum corneum [1]. Small amounts of cholesterol esters and
cholesterol sulfate may be present in stratum corneum. Representative structures
of all of the major lipids found in human stratum corneum are shown in Figure 1.
In addition to the extractable lipids, there are covalently bound lipids coating
the outer surface of the cornified envelope in epidermal stratum corneum
[42–44]. This consists mainly of ceramides. In porcine stratum corneum the
principal covalently bound lipid is CER OS derived from CER EOS [42, 44]. In
human stratum corneum, in addition to covalently bound CER OS, a second
more polar covalently bound ceramide was found [43]. This was later shown to
be CEROH [40]. Representative structures of CER OS and CER OH are
presented in Figure 2. In addition to ceramide, there are covalently bound -
hydroxy acids and free fatty acids in epidermal stratum corneum[44]. Small
proportions of covalently bound cholesterol and glucosylceramides have also
been reported [46].
5
“CONFIDENCE INTERVALS” FOR THE “TRUE”
LIPID COMPOSITION OF THE HUMAN SKIN
BARRIER
The outcome of experiments using mixtures of synthetic or extracted skin lipids
as models of skin barrier structural organization ultimately depends on the
chosen lipid composition. For experiments of kinds of, it is therefore a
prerequisite to have access to accurate and precise (within a few molar percent)
compositional data. It is quite evident from Tables 1–5 that there is no consensus
in the literature (within a few molar percent) with respect to the lipid
composition of the stratum corneum intercellular lipid matrix, and quantitative
information regarding the composition of the covalently bound lipids is very
limited (Table 5). In fact, from a biophysical point of view it is more of a horror
scenario, with reports in different studies of up to 8-fold differences (in wt %)
for single lipid classes (cf. Table 1), 3-fold differences (in wt %) for single
ceramide fractions (cf. Table 2), and 20-fold differences (in wt %) for single free
fatty acid fractions (cf. Table 3).
FIGURE 1 Representative structures of the lipids of human stratum corneum.

FIGURE 2 Representative structures of the covalently bound lipids from human stratum
corneum.
TABLE 1 General Lipid Composition of Human Stratum Corneum (wt of Total Extracted Stratum Corneum Lipid)a
aData in italics emphasize different results obtained with different preparation procedures.
bRates of cholestrol to caramide, the only lipid fractions that are free from contaminations.
cInvestigators used 24 wt% wax esters, 7 wt% squalene of total lipid fraction.
dInvestigators used 11 wt% wax esters, 21 wt% squalene, 5 wt% phospholipids of total lipid fraction.
eSample was extracted from cyanoacrylate forearm strippings.
f Sample was extracted from stratum corneum isolated from forearm skin biops. samples.
gThe reported ratio of cholesterol to cholesterol sulfate was 95.5.
hNo data, due to a fundamental difference in perception between different research groups.
“CONFIDENCE INTERVALS” FOR LIPID COMPOSITION?
87
TABLE 2 Ceramide Composition of Human Stratum Corneum as Determined by Thin-Layer Chromatography [wt of Total Ceramide (CER)
Fraction]
a
N-(Triacontanoyl- -O-linoleyl)-sphingosine.
b -(Stearoyl)-sphingosine.
N
c -(Stearoyl)-4-hydroxysphinganine.
N
d -(Triacontanoyl- -O-linoleyl)-6-hydroxysphingosine.
N
e -(2-Hydroxystearoyl)-sphingosine (AS)+N-(stearoyl)-6-hydroxysphingosine (NH).
N
f -(2-Hydroxystearoyl)-4-hydroxysphinganine.
N
g -(2-Hydroxystearoyl)-6-hydroxysphingosine extracted from cyanoacrylate forearm strippings.
N
hSample
TABLE 3 Free Fatty Acid Composition of Human Stratum Corneum as Determined by Various Gas Chromatographic Techniques (wt of Total
FFA Fraction)
aNo data recorded owing to a fundamental differences in preception between different research groups.
“CONFIDENCE INTERVALS” FOR LIPID COMPOSITION?
89
TABLE 4 Change in Lipid Composition with Depth in Human Stratum Corneum

Ratio of Stripped to nonstripped
Number of Technique FFA Cholestero Ceramide Chol/ Cer Study Ref.
Subjects l
7 LC/LSD 0.8 1.3 1.1 1.2 Norlén et 47
al., 1999
6 TLC 0.6 1.3 0.9 1.4 Bonté et 53
al., 1997
5 TLC 0.6 1.4 1.1 1.3 Weerheim 21
& Ponec,
2001
0.6 1.3 1.1 1.3 Median
(wt/wt)
0.6–0.8 1.3–1.4 0.9–1.1 1.2–1.4 Range (wt/
wt)
1.3 1.1 1.2 1.2 Max/Min
To our knowledge, the only studies to date that address, in a statistically

rigorous manner, the experimental and interindividual variation in total stratum
corneum lipid composition* and in the stratum corneum free fatty acid
composition, respectively, and the depth-dependent compositional changes in the
stratum corneum, are the studies by Norlén et al. [8, 47]. From these studies it is
evident that the interindividual variation in the skin barrier lipid composition
may be small—a few molar percent—in comparison to the experimental error
(including instrumental error, variation in lipid contamination, variation in lipid
extraction efficiency etc.), being one to two orders of magnitude larger [8, 47]. One
may therefore presume that the confused
TABLE 5 Composition of Covalently Bound Lipids in Human Epidermal Stratum

Corneum Determined by Thin-Layer Chromatography
Fraction of lipids (wt %)
Number of CER OS CEROH -Hydroxy Fatty acids Study Ref.
Subjects acids
5 53.3 24.8 9.4 12.7 Wertz et al., 43
1989
18 46.6 21.1 16.9 15.4 Paige et al., 45
1994
47–53 21–25 9–17 13–15 Median Range
*Covalently bound lipid fractions not included.

TABLE 6 “Confidence Intervals” for Nonpolar and Polar Lipid Fractions in Human
Stratum Corneuma
Nonpolar fraction
Alkanes [0–0]
Squalene [0–0]
Wax esters [0–0]
Cholesteryl esters [0–20]
Triacylglycerols [0–0]
Unsaturated FFA Free fatty acids [0–?]
Saturated FFA Free fatty acids [7–13]
Cholesterol [20–33]
Total ceramides [40–50]
N-(Triacontanoyl- -O- [6–12]b
linoleyl)-sphingosine
(EOS, ceramide 1)
N-(Stearoyl)-sphingosine [12–25]b
(NS, Ceramide 2)
N-(Stearoyl)-4- [11–34]b
hydroxysphinganine (NP,
Ceramide 3)
N-(Triacontanoyl- -O-
linoleyl)-6-
hydroxysphingosine
(EO, Ceramide 4) [4–9]b
N-(2-Hydroxystearoyl)-
sphingosine (AS,
Ceramide 5) +
N-(Stearoyl)-6- [17–27]b
hydroxysphingosine (NH)
N-(2-Hydroxystearoyl)-4-
hydroxysphinganine
(AP, Ceramide 6) [4–11]b
N-(2-Hydroxystearoyl)-6-
hydroxysphingosine
(AH, Ceramide 7) [10–27]b
Polar fraction
Total cerebrosides [0–1]
Total phospholipids [0–0]
Cholesterol sulfate [0–7]
*Does not include covalently bound lipids.
aNote that these “confidence intervals” are not statistical confidence intervals (Cl) but
speculations based on the arguments put forth by the authors in this chapter.
bRepresents weight percent of total ceramide fraction.
situation today with regard to the “true” lipid composition of the skin barrier is
less due to biological variables (i.e., interindividual variation, body site variation,
seasonal variation etc.) than to a certain lack of procedural rigor in experimental
(e.g., lack of reference to blanks, absence of reports of experimental errors) and
statistical methods (e.g., repeated use of the t test to compare the amounts of
different lipid classes expressed in percent of total).
As an additional complicating feature, skin lipid analysis is particularly prone
to contamination, since the total endogenous lipid amounts extracted from the
stratum corneum typically are very small. Also, there exist a large number of
contamination sources (e.g., sebum, subcutaneous fat, environmental
contamination sources, enumerated earlier in Sec. 3). Consequently,
triacylglycerols and wax esters almost certainly represent contaminants [7].
However, unsaturated medium-chain free fatty acids (C16:1, C18:1) are also
likely contamination candidates, since they are present in large quantities in the
environment [e.g., on dust particles, etc. (personal communication, Professor
Tomas Cronholm)] and the skin sebum (after partial hydrolysis of the
triacylglycerols) [48].
The cholesterol ester found among the stratum corneum lipids is mainly
cholesterol oleate [7], thought to be mainly of epidermal origin. However,
cholesterol oleate is not a membrane-forming lipid, and it has been proposed that
it separates into a liquid phase within the intercellular spaces [35]. In this view, it
would not be a part of the lamellar membrane system. Likewise, cholesterol
sulfate is not delivered to the intercellular space along with the other lipids via
the lamellar granules [6]. It has been suggested that this lipid may be
preferentially associated with desmosomes, and recent observations support this
suggestion [50].
In 1987 Wertz et al. [7] analyzed the lipid composition of epidermal cysts,
containing by comparison enormous amounts of stratum corneum lipids, thereby
minimizing the effect of contamination. Two years later a result very close to
that of Wertz et al. [7] was obtained when quantitative HPLC-LSD was applied
to lipids extracted from the forearm of 22 healthy subjects after 15 tape
strippings to remove two-thirds of the stratum corneum thickness [47]. The
present authors therefore propose that the population mean total (non polar+polar
fraction) lipid composition* of human stratum corneum (in wt %) can be found
within the “confidence intervals” given in Table 6.
The lipid, ceramide, and free fatty acid (FFA) data presented in Tables 1–5 are
taken from the work of Wertz and Norlén just cited, as well as other papers cited
either and some [51–62] that are not discussed in the text.
ACKNOWLEDGMENTS
The present work was made possible by the generous support from the Wenner-
Gren Foundations (LN).
REFERENCES
1. Gray GM, Yardley HJ. Different populations of pig epidermal cells: isolation and
lipid composition. J. Lipid Res. 1975; 16:441–447.
2. Landmann L. The epidermal permeability barrier. Anat. Embryol. 1988; 178:1–13.
3. Landmann, L. Epidermal permeability barrier: transformation of lamellar granule-
disks into intercellular sheets by a membrane-fusion process, a freezefracture
study. J. Invest. Dermatol. 1986; 87(2):202–209.
4. Freinkel RK., Traczyk TN. Lipid composition and acid hydrolase content of
lamellar granules of fetal rat epidermis. J. Invest. Dermatol. 1985; 85:295–298.
5. Grayson S., Johnson-Winegar AG., Wintraub BU., Isseroff RR., Epstein EH., Elias
PM. Lamellar body-enriched fractions from neonatal mice: preparative techniques
and partial characterization. J. Invest. Dermatol. 1985; 85:289–294.
6. Madison KC, Sando GN., Howard EJ.,True CA., Gilbert D., Swartzendruber DC.,
Wertz PW. Lamellar granule biogenesis: a role for ceramide glucosyltransferase,
lysosomal enzyme transport, and the Golgi. J. Invest. Dermatol. Symp. Proc. 1998;
3:80–86.
7. Wertz PW., Swartzendruber DC, Kathi CM., Downing DT. Composition and
morphology of epidermal cyst lipids. J. Invest. Dermatol. 1987; 89:419–424.
8. Norlén L., Nicander L, Lundsjö A., Cronholm T., Forslind B. A new HPLC-based
method for the quantitative analysis of inner stratum corneum lipids with special
reference to the free fatty acid fraction. Arch. Dermtol Res. 1998; 290 : 508–516.
9. Madison KC., Swartzendruber DC., Wertz PW., Downing DT. Murine keratinocyte
cultures grown at the air/medium interface synthesize stratum corneum lipids and
“recycle” linoleate during differentiation. J. Invest. Dermatol. 1989; 93:10–17.
10. Madison KC, Swartzendruber DC., Wertz PW., Downing DT. Presence of intact
intercellular lamellae in the upper layers of the stratum corneum. J. Invest.
Dermatol. 1987; 88:714–718.
11. Forslind B. A domain mosaic model of the skin barrier. Acta Dermtol Venereol.
(Stockh.) 1994; 74:1–6.
12. Norlén, L. Skin barrier formation: the membrane folding model. J. Invest.
Dermatol. 2001; 117(4):823–829.
13. Norlén L. Skin barrier structure and function: the single gel-phase model. J. Invest.
Dermatol. 2001; 117(4):830–836.
14. Norlén L. Does the single gel phase exist in stratum corneum? Reply. J. Invest.
Dermatol. 2002; 118(5): 899–901.
15. Cremesti AE., Fischl AS. Current methods for the identification and quantitation of
ceramides: an overview. Lipids 2000; 35:937–945.
16. Myher JJ., Kuksis A. General strategies in chromatographic analysis of lipids. J.
Chromatogr. B: Biomed. Appl. 1995; 671:3–33.
17. Hoving EB. Chromatographic methods in the analysis of cholesterol and related
lipids. J. Chromatogr. B: Biomed. Appl. 1995; 671:341–362.
18. Downing DT. Photodensitometry in the thin-layer chromatographic analysis of
neutral lipids. J. Chromatogr. 1968; 38:91–99.
19. Touchstone JC., Chen JC., Beaver KM. Improved separation of phospholipids in
thin layer chromatography. Lipids 1980; 15:61–62.
20. Poole CF, Poole SK. Progress in densitometry for quantitation in planar
chromatography. J. Chromatogr. 1989; 492:539–584.
21. Weerheim A., Ponec M. Determination of stratum corneum lipid profile by tape
stripping in combination with high-performance thin-layer chromatography. Arch.
Dermatol. Res. 2001; 293:191–199.
22. Motta S., Monti M., Sesana S., Ghidoni R., Caputo R. Abnormalities of water
barrier function in psoriasis. Role of ceramide fractions. Arch. Dermatol. 1994;
130:452–456.
24. Okumura K., Hayashi K., Morishima I., Murase K., Matsui H., Toki Y, Ito T.
Simultaneous quantiyation of ceramides and 1,2-diacylglycerol in tissues by
latroscan thin-layer chromatography-flame-ionization detection. Lipids 1998; 33:
529–532.
25. Shanta NC. Thin-layer chromatography-flame ionization detection latroscan system.
J.Chromatogr. 1992; 624:21–35.
26. Brown BE., Williams ML., Elias PM. Stratum corneum lipid abnormalities in
ichthyosis. Detection by a new lipid microanalytical method. Arch. Dermatol. 1984;
120:204–209.
27. Iatroscan Symposium (anonymous). Papers from the symposium on Analysis by
latroscan TLC/FID System presented at the 75th annual meeting of the American
Oil Chemists Society, Dallas, TEX, April 1984. Lipids 1985; 20:501–560.
28. Christie WW. High-Performance Liquid Chromatography and Lipids. Pergamon
Press, Oxford, 1987.
29. Christie WW. Gas Liquid Chromatography and Lipids. Oily Press, Ayr, Scotland,
1989.
30. Evershed RP. In: Hamilton RJ, Hamilton S (eds) Lipid Analysis. A Practical
Approach. Oxford University Press, Oxford, 1992.
31. Ackman RG., Sipos JC. Application of specific response factors in the gas
chromatography analysis of methyl esters of fatty acids with flame ionisation
detectors. J.Am. Oil Chem. Soc. 1964; 41:377–378.
32. Bortz JT., Wertz PW., Downing DT. On the origin of alkanes found in human skin
surface lipids. J. Invest. Dermatol. 1989; 93:723–727.
33. Wertz PW., Michniak BB. Sebum. In: Cosmeceuticals, P.Elsner, HI. Maibach
(eds.) Marcel Dekker., New York, 2000; 45–56.
34. Wertz PW., Downing DT. Ceramides of pig epidermis: structure determination. J.
Lipid Res. 1983; 24:759–765.
35. Wertz PW. Kremer M. Squier CA. Comparison of lipids from epidermal and palatal
stratum corneum. J. Invest. Dermatol. 1992; 98:375–378.
36. McIntosh TJ., Stewart ME. Downing DT. X-ray diffraction analysis of isolated skin
lipids: reconstitution of intercellular domains. Biochemistry 1996; 35:3649–3653.
37. Bouwstra JA., Gooris GS., Dubbelaar FER., Weerheim AM., Izerman AP. Role of
ceramide 1 in the molecular organization of the stratum corneum lipids. J. Lipid Res.
1998; 39:186–196.
38. Bouwstra JA., Dubbelaar FE., Gooris GS., Ponec M. The lipid organization of the
skin barrier. Acta DermatoVenereol. 2000; suppl 208:23–30.
39. Wertz PW., Miethke MC., Long SA., Strauss JS., Downing DT. The composition of
the ceramides from human stratum corneum and from comedones. J. Invest.
Dermatol. 1985; 84:410–412.
40. Robson KJ., Stewart ME., Michelsen S., Lazo ND., Downing DT. 6-Hydroxy-4-
sphingenine in human epidermal ceramides. J. Lipid Res. 1994; 35:2060–2068.
41. Stewart ME., Downing DT. A new 6-hydroxy-4-sphingenine-containing ceramide

in human skin. J. Lipid Res. 1999; 40:1434–1439.
42. Wertz PW., Downing DT. Covalently bound -hydroxyacylsphingosine in the
stratum corneum. Biochim. Biophys. Acta 1987; 917:108–111.
43. Wertz PW., Madison KC., Downing DT. Covalently bound lipids of human stratum
corneum. J. Invest. Dermatol. 1989; 91:109–111.
44. Chang F., Swartzendruber DC., Wertz PW., Squier CA. Covalently bound lipids in
keratinizing epithelia. Biochim. Biophys. Acta 1993; 1150: 98–102.
45. Paige DG., Morse-Fisher N., Harper JI. Quantification of stratum corneum
ceramides and lipid envelope ceramides in hereditary ichthyoses. B.J. Dermatol.
1994; 131:23–27.
46. Serizawa S., Ito M., Hamanaka S., Otsuka F. Bound lipids liberated by alkaline
hydrolysis after exhaustive extraction of pulverized clavus. Arch. Dermatol. Res.
1993; 284:472–475.
47. Norlén L., Nicander I., Lundh-Rozell B., Ollmar S., Forslind, B. Inter and intra
individual differences in human stratum corneum lipid content related to physical
parameters of skin barrier function in-vivo. J. Invest. Dermatol. 1999; 112(1):72–
77.
48. Stewart ME., Downing DT. Chemistry and function of mammalian sebaceous
lipids. In: PM, Elias (Ed.) Advances in Lipid Research—Skin Lipids. Vol. 24,
Academic Press, San Diego, CA; 1991; 263–301.
50. Li S.,Wertz PW. Association of cholesterol sulphate with desmosomes. (abstr) J.
Dent. Res. 2002 (In Press).
51. Lampe M A., Burlingame AL., Whitney J., Williams ML., Brown BE., Roitman
E., Elias PM. Human stratum corneum lipids: characterization and regional
variations. J. Lipid Res. 1983; 24:120–130.
52. Lévêque J-L., de Rigal J., Saint-Léger D., Billy, D. How does sodium lauryl sulfate
alter the skin barrier function in man? A multiparametric approach. Skin
Pharmacol. 1993(6):111–115.
53. Bonté F., Saunois A., Pinguet P., Meybeck A. Existence of a lipid gradient in the
upper stratum corneum and its possible biological significance. Arch. Dermatol.
Res. 1997; 289:78–82.
54. Bonté F., Pinguet P., Saunois A., Meybeck A., Beguin S., Ollivon M., Lesieur S.
Thermotropic phase behaviour of in-vivo extracted human stratum corneum lipids.
Lipids 1997; 32(6):653–660.
55. Yamamoto A., Serizawa S., Ito M., Sato Y.Stratum corneum lipid anomalities in
atopic dermatitis. Arch. Dermatol. Res. 1991; 283:219–223.
56. Di Nardo A., Wertz P., Gianetti A., Seidenari S. Ceramide and cholesterol
composition of the skin of patients with atopic dermatitis. Arch. Dermatol
Venereol (Stockh) 1998; 78:27–30.
57. Bleck O., Abeck D., Ring J., Hoppe U., Vietzke J-P, Wolber R., Brandt O.,
Schreiner V. Two ceramide subfractions detectable in Cer (AS) position by HPTLC
in skin surface lipids of non-lesional skin of atopic eczema. J. Invest. Dermatol.
1999; 113(6):894–900.
58. Rogers J., Harding C., Mayo A., Banks J., Rawlings A. Stratum corneum lipids: the
effect of ageing and the seasons. Arch. Dermatol. Res. 1996; 288:765–770.
59. Long SA., Wertz PW., Strauss JS., Downing DT. Human stratum corneum polar
lipids and desquamation. Arch. Dermatol. Res. 1985; 277:284–287.
60. Imokawa G., Abe A., Jin K., Higaki Y., Kawashima M., Hidano A. Decreased
level of ceramides in stratum corneum of atopic dermatitis: an etiologic factor in
atopic dry skin. J. Invest. Dermatol. 1991; 96(4):523–526.
61. Matsumoto M., Umemoto N., Sugiura H., Uehara M. Difference in ceramide
composition between “dry” and “normal” skin in patients with atopic dermatitis.
Arch. Dermatol. Venereol (Stockh) 1998; 79:246–247.
62. Nicollier M., Massengo T., Rémy-Martin J-P, Laurent R., Adessi G-L. Free fatty
acids and fatty acids of triacylglycerols in normal and hyperkeratotic human
stratum corneum. J. Invest. Dermatol. 1986; 87(1):68–71.
6
Stratum Corneum Lipid Organization In Vitro
and In Vivo as Assessed by Diffraction
Methods
Gonneke S.K.Pilgram
Leiden University Medical Center, Leiden, The Netherlands
Joke A.Bouwstra
Amsterdam Center for Drug Research, Leiden University, Leiden,
The Netherlands
The epidermis of mammalian skin is a stratified squamous epithelium that

provides the body with a barrier against influences from the environment.
Important features of this barrier, which is located in the stratum corneum (SC),
include protection against desiccation and the penetration of microbes and
harmful agents. The outermost nonviable layer of the epidermis, the SC consists
of flat, protein-rich corneocytes embedded in an intercellular lipid matrix. It has
been shown that most agents that penetrate the skin need to pass this intercellular
lipid matrix, since these lipids form an almost continuous domain in the SC [1].
Consequently, the intercellular lipid matrix, is an important topic in studies on
(trans) dermal drug delivery systems [2–6], barrier function, desquamation,
diseased skin, and reconstructed epidermis [7–11].
This chapter describes two complementary techniques, (wide-angle and small-
angle) X-ray diffraction [W(S)AXD] and electron diffraction (ED), which
provide insight into the lamellar and lateral organization of lipid alkyl chains
within the SC intercellular domains and SC lipid models. We demonstrate that
these techniques can be used to obtain valuable information, which contributes to
our understanding of the relationship between the composition and organization
of SC lipids. This kind of information is crucial for elucidating the barrier
function of human skin.
1
PRELIMINARY CONSIDERATIONS
1.1
Stratum Corneum Lipid Composition
Lamellar bodies contain precursor lipids for the intercellular matrix, as well as
different kinds of catabolic enzyme. After extrusion of the lamellar bodies at the
98 PILGRAM AND BOUWSTRA
stratum granulosum-SC interface, hydrolysis of the precursor lipids converts the

glycosphingolipids and sphingomyelin into ceramides (CER), and phospholipids
into free fatty acids (FFA) (reviewed in Ref. [12]). Thus, in contrast to the viable
layers of the epidermis, where phospholipids are the major components of the
cell membranes, the ceramides in SC become the major constituents of the
intercellular domains [13, 14] among other lipids like long-chain FFA,
cholesterol (CHOL), and cholesterol sulfate [15]. In addition to its exceptional
lipid composition, the structure of the CER is unusual for membrane-forming
lipids, because the SC CER contain very long acyl chains and small polar head
groups, which bind water poorly. The main constituents of the intercellular lipids
are described next.
1.1.1
Ceramides
Figure 1 depicts the different classes of CER that are present in porcine and
human SC [4, 14]. Eight classes of CER have been detected in human SC [16, 17].
These subtypes differ in molecular structure and polarity and can be identified
using high-performance thin-layer chromatography (HPTLC). Ceramides are
composed of a sphingosine, phytosphingosine, or 6-hydroxy-sphingosine base
with variations in position and number of hydroxyl groups, double bonds, and
chain length [15]. Furthermore, long-chain nonhydroxy or -hydroxy fatty acids
are linked to the (phyto) sphingosine and 6-hydroxy-sphingosine bases through
an amide bonding. The CER 1 (as well as CER 4 in humans) is especially unique
in that it contains an additional fatty acid (linoleic acid is the most abundant
one), which is esterified with the hydroxyl group of an amide-linked long-chain
-hydroxy acid [18]. As a consequence, an unusual long molecule with a total
chain length of up to 50 carbons may be formed. It is suggested that CER 1 has a
specific function in the formation of the lipid envelope and the lamellar
organization of the intercellular SC lipids [14–19].
1.1.2
Free Fatty Acids
The hydrocarbon backbone of FFA in the intercellular matrix of the SC is
predominantly long chain and saturated [20]. However, the relative amounts of
FFA differ between human (prominent chain lengths are 11 % C22, 39% C24,
and 23% C26) and porcine SC (31 % C22, 25% C24) [15, 20]. Oleic acid (5.7%
C18:1) and linoleic acid (1.4% C18:2) are the only unsaturated FFA detected in
the SC, although it has been suggested that these FFA should be regarded as
contamination from sebaceous lipids or the environment [20]. The FFA in sebum,
which are derived from the triglycerides, have shorter hydrocarbon chains and
are mainly unsaturated (C16:1 and C18:1). It has been suggested that pathways
exist for the degradation and/or recycling of unsaturated fatty acids, since the
STRATUM CORNEUM LIPID ORGANIZATION 99
FIGURE 1 Molecular structures of eight subclasses of ceramides found in human SC and

of six subclasses of pig ceramides. Ceramides 1, 2, 3, and 6 are structurally similar. Note
that CER 4 in humans resembles CER 1, while this subtype is absent in pig ceramides.
FFA composition of the SC differs from that of the underlying layers.

Furthermore, these long-chain FFA play an important role in SC barrier function,
as will be described in this chapter.
1.1.3
Cholesterol
Of the sterols present in human SC, CHOL predominates, although significant
levels of cholesteryl esters and cholesterol sulfate have also been detected.
Cholesterol is the only major lipid class that is present in both the plasma
membranes and the intercellular lamellae. A specific physical feature of CHOL
is the planar steroid ring, which is a conformationally rigid structure. When
present in large amounts in phospholipid membranes, CHOL increases the
permeability barrier by introducing conformational ordering of the lipid chains,
while retaining the membrane fluid [21–23]. In SC, the CHOL behavior is very
complex, as well (see later).
1.2
Stratum Corneum Lipid Organization
Amphiphilic lipids have a hydrophilic head group and a hydrophobic
hydrocarbon chain, which make it possible for lipids to form three-dimensional
structures such as micelles and hexagonal, cubic, and lamellar phases. The lipid
organization in lamellar phases is similar to that of biological membranes. In
these structures, the lipids are held together by forces including van der Waals,
electrostatic, hydrophobic, and hydrogen-bonding interactions. The packing
properties of the amphiphilic lipids depend on the size of their head group, their
acyl chain length, and the degree of saturation. External factors that influence the
packing density include pH, temperature, and pressure. In the solid state, the
lateral packing of lipids in lamellae is either in a hexagonal (gel), orthorhombic
(crystalline), or a triclinic subcell (Fig. 3). Furthermore, the lipids can form a
liquid lateral packing as well.
In taking into account factors influencing the lipid organization, one can
speculate on the marked differences between the organization of phospholipid
membranes, assembled from short-chain (C16–C18), polar, often unsaturated
lipids and SC lipid lamellae, which contain relatively long-chain (C22–C26), non
polar, often saturated lipids. In their lateral packing and phase behavior, these
distinct lipid systems differ considerably from phospholipid membranes. For
example, the phase behavior of CHOL in phospholipid membranes is very
complex and dualistic in that it reduces the lipid mobility in a liquid phase as it
reduces the molecular head group area through its plane surface, whereas it
increases the chain mobility in gel phases [24]. A further increase in CHOL
content in gel phases even induces a transition from a gel to liquid phase in
phospholipid systems. In contrast, when CHOL levels are increased in mixtures
prepared with CER, no transition from a gel to a liquid phase has ever been
observed. This immediately indicates the uniqueness of the intercellular lipids, of
which the structure, composition, and organization are crucial for the specific
barrier properties of the SC.
This chapter begins by briefly describing the X-ray diffraction and ED
methods. Section 3 treats the lipid organization in intact human SC. To examine
the role of individual lipid classes in the SC section 4, describes mixtures
prepared with CHOL, FFA, and CER isolated from human and pig SC. Finally,
the information obtained in these studies has been used to construct a model that
describes the molecular organization in the SC. This model, referred to as the
sandwich model, is discussed in Section 5.
2
METHODS
2.1
X-Ray Diffraction
2.1.1
General Description
In general, an incident X-ray beam interacts with the electrons of the molecules
in a sample and the scattered beam is measured by an electronic detector, an
image plate, or a photographic film. In fact, the detection system measures the
scattered intensity as a function of the scattering angle , (Fig. 2 A). The
scattered intensity as a function of the scattering angle is often referred to as the
FIGURE 2 (A) Schematic presentation of SAXD and WAXD: X-rays are produced by a
source and scattered by a sample. At the detection plane the scattered intensity is
measured as a function of . WAXD provides information about the smaller structural
units in the sample such as the lateral packing, while SAXD provides information about
the larger structural units in the sample such as the lamellar ordering. (B) When the
difference in X-ray path length between two successive planes equals an integer (n) times
the wavelength ( ), the X-rays from successive planes are in phase. In accordance with
Bragg’s law, in-phase X-rays amplify and result in a maximum of intensity at the plane of
detection.
diffraction pattern or diffraction curve. The diffraction pattern is determined by
the electron density of the atoms and by the position of the atoms in the sample.
The maximum possible amplitude of the scattered X-rays of a single isolated
atom is determined by the oscillation of the electrons of an atom. The
oscillations are mainly determined by the interactions between the electrons and
the primary X-ray beam. However, the diffraction pattern does not depend on the
maximum amplitudes from the individual atoms alone; it depends also on the
interference between the X-rays scattered from the various atoms. This
interference is determined by the positions of the individual atoms. The positions
of the atoms or molecules are often described by the unit cell, which is the
smallest structural unit from which the integral structure can be generated.
In a well-defined structure in which the molecules are located at fixed position,
the lattice can be described by several series of parallel lattice planes oriented in
various directions. These lattice planes act as partially reflecting mirrors. The
reflected X-rays interfere and a maximum intensity is obtained when the difference
in path length between the X-rays reflected from two successive planes is an
integer multiplied by the wavelength (see Fig. 2b). This is the well-known
Bragg’s law, n =2d sin , in which is the scattering angle, the wavelength
(typically 0.1–0.15 nm), and d the distance between two planes, or alternatively
(1)
FIGURE 3 (A) Small-angle X-ray diffraction. The intensity of the scattered X-rays is
plotted as function of Q. The diffraction pattern of a lamellar phase consists of a series of
peaks, referred to as the first order located at Q1, second order located at Q2, third order
located at Q3, and so on. Since the distance between the sequential peaks is equal, the
peak positions are related as Q3=3Q1, Q2=2Q1, and so on. From the positions of these
peaks, the periodicity dA (in the three dimensional structure in the z direction) of the
lamellar phase can be calculated directly by da=2 /Q1=4 /Q2=6 /Q3, and so on. When the
repeat distance of the lamellar phase is larger, the distance between the sequential peaks is
smaller (compare dA (LP) with dB (SP)). If two lamellar phases (LP + SP) are present in
the sample, the diffraction peaks of the two phases are additive. This often results in a
formation of a broader peak with a shoulder (see, e.g., Fig. 7). (B) The orientation of the
lipid lamellae with respect to the virtual axes x, y and z. (C) Wide-angle X-ray diffraction.
Schematic presentation of the positions of the alkyl chains in liquid, hexagonal, and
orthorhombic phases parallel to the basal plane (i.e., in x-y direction perpendicular to the z
direction) of the lamellae and their corresponding diffraction patterns. Owing to a large
sample size (typically a few milligrams), lattices in different orientation are exposed to the
X-ray beam. This results in diffraction patterns that consist of a series of rings. In a liquid
phase (high permeability), the distances between the hydrocarbon chains are not very well
defined, resulting in a broad reflection at approximately 0.46 nm. In a hexagonal packing
(medium permeability), the hydrocarbon chains of the lipids are equally distributed in the
structure at interchain distances of 0.48 nm (spacing 0.41 nm). This results in a strong
reflection (ring) at approximately 0.41 nm spacing. The orthorhombic phase (low
permeability) is a very dense structure, of which the hydrocarbon chains are not equally
distributed in the lattice. This results in a diffraction pattern with two rings at 0.41 and at
0.37 nm, respectively. Furthermore, the Miller indices (h k l) of the lattice planes are
shown. As the lateral lipid organization is studied, only the two-dimensional ordering of
the lipids is depicted and “l” of the Miller indices is zero, because the three-dimensional
structure representing the lamellar organization is not shown. (D) Electron diffraction.
Schematic representation of ED patterns obtained when a small area is selected for
diffraction. The reflections characteristic of orthorhombic and hexagonal packing are
separated in spots or arcs. When larger areas are selected, however, ring patterns will be
obtained similar to those shown in (C). For each set of parallel lattice planes, two opposite
reflections appear perpendicularly oriented with respect to these repetitive planes.
By measuring the position of the maximum intensity in the detection plane as

function of the scattering angle , the distance d can be calculated. Note that a
reciprocal relationship exists between and d.
2.1.2
Small Angle X-Ray Diffraction
The diffraction pattern of a single membrane can be calculated from the electron
density distribution (x) in the membrane by Fourier transformations, in which x
is the spatial coordinate perpendicular to the basal plane of the membrane and Q
is the reciprocal space vector (Q=4 sin / ). The Fourier transform defines both
the amplitude and the phase of the scattered X-ray. In case of a membrane system
the Fourier transform (Fm) can be written as follows:
(2)
where d is the thickness of the membrane or the length of the repeating unit. In
general the Fourier transform consists of a real and an imaginary part. To relate
the Fourier transform to the information obtained in reciprocal space that is on the
detection plane, we multiply the Fourier transform by its complex conjugate Fm
(Q)*. Therefore the intensity I(Q) can be described as follows:
(3)
Unfortunately, multiplication of the complex conjugate results in loss of the

phase information. This makes the inverse procedure, in which the electron
density is calculated from the experimental diffraction curve, nontrivial, a
phenomenon known in the literature as the phase problem. In
symmetrical structures, often encountered in biological membranes, the
imaginary part of the Fourier transform vanishes when one is calculating I(Q).
Then the phases are limited to 0 (the Fourier transform is positive) or , which
means that the sign of the Fourier transform is negative. In the case of
asymmetric structures, the Fourier transform consists of a real and an imaginary
part, and the phases can adopt all values between 0 and 2 . One method dealing
with the phase problem [25] focuses on swelling of the bilayers, assuming that
the bilayer electron density is not dependent on the amount of water between the
bilayers. Another method is heavy-atom labeling, in which heavy atoms are fixed
on a specific position in the bilayers, assuming that these heavy atoms do not
change the structure upon being incorporated in the membrane. In dealing with
asymmetric membranes, it is in general not possible to calculate the electron
density of a membrane from diffraction data without prior knowledge of the
structure.
When the sample consists of stacked membranes forming a multilamellar

array, a diffraction pattern with a discrete number of intensities is expected. The
series of peaks often is referred to as first-, second-, and so on order peaks (n=1,
2, …). From the position of these peaks, one can use Bragg’s law to calculate
directly the repeat distance of the lamellar phase, namely, d=2 n/Qn, in which n
is the order of the diffraction peak.
2.1.3
The Lorenz Factor and Disorder in a Lattice
The Lorenz factor depends on the orientation of the membrane stacks. In Eqs. (1)
to (3), it is assumed that all the membranes are oriented in the same direction. In
this situation the diffraction in the plane of detection consists of a series of spots.
However, if the stacked bilayers are not perfectly oriented, the radial spot width
becomes wider and subsequently the spots are turned into arcs. A further
increase in the orientational disorder of the stacks will widen the radial intensity
distribution further, and finally the intensity is distributed in a series of spherical
shells instead of spots. This pattern also has consequences for the relative
intensities as function of Q. If the sample consists of a series of randomly
oriented stacks, the scattered intensity is spread over a larger shell at higher Q
values. This means that to use a linear detection system, one must multiply the
measured intensity by Q2, the so-called Lorenz factor, to fit the calculated
intensities with the experimental ones.
Disorder due to thermal motion is often referred to as disorder of the first kind
[25]. An increase in the thermal motion results in a distribution of the electrons
over a larger space than in the absence of thermal motion. Another type of
disorder encountered in membrane stacks is a slight variation in repeat distance.
In this case the actual distance is mainly determined by the adjacent membranes
and not by the integral membrane system. This disorder is referred to as disorder
of the second kind. A third type of imperfection of a membrane system is the
presence of undulations. This results in a variation in width between adjacent
membranes. Each type of disorder encountered in the lattice will affect the
diffraction pattern, differently.
2.1.4
Wide-Angle X-Ray Diffraction
Wide-angle X-ray diffraction (WAXD) patterns of membranes provide
information on the smaller structural units in the sample, the lateral packing of
the lipids. Dealing with a single crystal that is ordered in three dimensions, the
WAXD pattern consists of series of spots, on well-defined positions. From the
positions of these spots, the lattice dimensions can be calculated, while the
intensity of the spots provides information about the localization of the individual
atoms. However, if the sample contains several crystals, the orientations of
which are slightly different, either the spots widen radially and turn into arcs or
separate additional spots appear. If the sample contains a large number of small
crystals randomly oriented, the arcs turn into rings (Fig. 3C). The position of the
rings or spots depends on the crystal lattice. In the 1970s Abrahamsson et al. [26]
published an excellent review of the different unit cells encountered in lipid
systems.
If the diffraction pattern consists of a series of rings, it is difficult to assign the
position of the rings to a unique lattice, since the information in the radial
direction has disappeared, unlike the case of the diffraction pattern of a single
crystal. Since in an ED experiment the area exposed to the primary beam is much
smaller, (as described shortly), the ED pattern is more likely to display
diffraction spots than rings. This facilitates the assignment to a particular lattice
of the pattern obtained. Only in exceptional cases can diffraction rings be
assigned to a particular sublattice. The most common lateral phases present in
the SC lipid organization are the liquid, the hexagonal, and the orthorhombic
subphase (Fig. 3C). The ED patterns of these phases are discussed next.
2.2
Electron Diffraction
The principles of ED are similar to those of X-ray diffraction. For completeness,
however, this technique is also described in some detail. The electron beam in a
transmission electron microscope (TEM) can be represented as parallel wave
planes with a certain wavelength, which depends on the acceleration voltage of
the electron microscope ( =0.0037 nm at 100 kV). The wavelength in an ED
experiment is much smaller than that used in work with X-rays. When the
electron beam passes through a sample, the electron waves will be scattered in
all directions by the atoms (scattering centers), and thus the scattering increases
with the atomic number and with sample thickness. In contrast to X-rays,
electrons that interfere with the atomic nuclei (elastic scattering) are most
important for image formation, while electrons that were inelastically scattered
upon interaction with electrons of the atoms tend to decrease the resolution. So
the postspecimen wave front differs from the prespecimen wave front and
contains information about the sample, as with X-rays.
For imaging, postspecimen lenses (including the electromagnetic objective
lens) of the TEM focus the scattered waves in such a way that electrons leaving
the sample from a certain point converge in the image plane (Fig. 4). The
ensemble of lenses in this setting results in an enlarged image displayed on the
fluorescence screen. In the diffraction mode of the TEM, the postspecimen
lenses have another setting that permits the back focal plane of the objective lens
to be imaged on the fluorescence screen. In this setting of the lenses, the image is
the diffraction pattern (Fig. 4). If a sample is crystalline, the ED pattern will
contain relatively sharp diffraction maxima, whereas in an image the same
electron flux is spread over the whole screen. Therefore, a much lower electron
FIGURE 4 Schematic representation of a projection of an object with spacing d in the

image plane and, simultaneously, an ED pattern formed in the back of the focal plane of
the objective lens. At n=0, which is the zero-order reflection maximum, forward-scattered
electrons are focused, while at the nth-order reflection maximum electrons converge with
a path length difference of n and diffraction angles . (Adapted from Ref. 27.)
dose (dose rate approximately 10 e /nm2·sec) can be used for the recording of an
ED pattern, which is advantageous for radiation-sensitive biological specimens,
such as SC lipid lamellae. To further minimize radiation damage, samples were
studied at 170°C.
The scattering of an electron wave by a row of equally spaced atoms will lead
to (constructive and destructive) interference depending on the scattering angle,
similar to the situation explained for X-rays. Constructive interference occurs for
successive path differences of multiples of n according to Bragg’s law n =2d
sin for ED. However, the diffracted wave is very small (sin ), which leads
to a simplification of Bragg’s law to Rd= L. In this equation R is the radius, the
distance from a reflection to the central spot, and L the camera length. For
calibration of the constant factor L, the diffraction pattern of a crystal with well-
defined lattice spacings can be used (e.g., the powder ED pattern of gold).
According to the formula, there is a reciprocal relationship between the spacing
and radius (determined by ). Thus an increase in the spacing results in a
decrease of the radius in the diffraction pattern. Furthermore, when decreases
(higher accelerating voltage) the radius decreases, as well.
Electron diffraction patterns can be described in terms of the reflection of
electrons by the lattice planes of the crystal (in two and three dimensions). Also
in this case electrons reflected by parallel sets of planes (with a certain spacing)
produce constructive interference and diffraction maxima provided the Bragg
condition is satisfied. The directions in a crystal and specific lattice planes can be
specified by Miller indices (h k l). Calculation of the positions for diffraction

maxima using these indices yield the same results obtained when the diffraction
grating model is used (for more details, see Ref. [27]).
As already mentioned, the main lateral phases in SC are orthorhombic,
hexagonal, and liquid. The liquid phase is characterized by abroad reflection
centered at 0.46 nm. We used the following formula for the orthorhombic system
and hexagonal system
to calculate the positions of the reflections of the (110), (200), (020) planes of the
orthorhombic lattice and the (110), (100) and (010) planes of the hexagonal
lattice. These lattice planes are shown in Figure 3C.
Since biological specimens often consist of atoms of low atomic number,
higher order reflections will mostly be absent because their intensity will be too
low. Furthermore, buckling of the specimen (resulting in a broadening of the
reflections, as described in the preceding section) and the presence of
inelastically scattered electrons (which proportionately increase with sample
thickness) may complicate the detection of the relatively faint, higher order
diffraction maxima. For the recording of ED patterns from SC lipids, we will
have to deal with these difficulties. However, there are some important
advantages in comparison to WAXD, which make ED a complementary
technique. First of all in vivo skin can be studied (see Sec. 2.4) with ED, whereas
this is not possible using WAXD. Furthermore, the smaller sample sizes allow
one to study the SC as a function of depth (see also Sec. 2.4). And finally, as
briefly discussed earlier, an ED pattern of an area as small as ~ 1 µm2 can be
obtained, which promotes the appearance of separate spots or arcs instead of
rings, thus assisting the researcher, to distinguish between the presence of an
orthorhombic lattice alone and the simultaneous presence of the hexagonal and
orthorhombic packing.
2.3
Extraction, Separation, and Identification of Lipids from
Stratum Corneum
2.3.1
Separation and Identification
Epidermal lipids are extracted by using the method of Bligh and Dyer [28], and
the extracts dissolved in chloroform-methanol (2:1, v/v) and stored at 20°C under
nitrogen until use. Subsequently, the extracted lipids are applied on a Silicagel 60
(Merck) column. The various lipid classes are eluted sequentially by using
various solvent mixtures [29]. The eluted lipids are collected in either 10 mL
fractions or 3 mL fractions, depending on whether ceramides are expected to be
present. The lipid composition of individual fractions is established by one-
dimensional high-performance thin-layer chromatography, as described before
[29]. For quantification, authentic standards (Sigma) are run in parallel,
consisting of 0.1 mg/mL for each of the components CHOL, bovine CER 3, and
FFA. After charring, a photodensitometer with automatic peak integration
(Desaga, Germany) is used to perform the quantification.
2.3.2
Preparation of Lipid Mixtures
The ceramides and cholesterol are mixed in various molar ratios, using a mean
ceramide molar weight of 700. For calculation of the mean ceramide molecular
weight, data on the ceramide composition and alkyl chain length distributions
[14] are used. Approximately 2 mg of lipids is solubilized in 80 µL of
chloroform-methanol (2:1) at the desired composition and applied with a sample
applicator (Camag Linomat IV) to mica at a very low rate (4.2 mL/min) under a
stream of nitrogen. The applied lipid mixtures are covered with 1 to 2 mL buffer
at the desired pH and kept under nitrogen. The pH of the skin surface [30] or the
viable epidermis (pH 5 or 7.4 respectively) is often chosen. To reach
homogeneous mixing of various lipid fractions, the lipids applied to mica are first
heated to 60 to 80°C (depending on the lipid mixtures) and kept at this temperature
for 2 to 5 min. Subsequently, the samples are quenched with dry ice. Then the
samples are placed in a small copper sample holder, after which at least 10
freeze-thaw cycles are carried out between —20°C and room temperature.
The preparation of samples for ED differs only with respect to the sample
holder and the amount of lipids required. Approximately 25 µg of a lipid mixture
is nebulized onto a 400-mesh copper grid (either plain or covered with a thin
carbon film). After the freeze-drying procedure, the samples are stored under
gaseous nitrogen at 20°C until use. Then the samples are either studied in the
TEM at room temperature or at 170°C by plunging them into ethane cooled by
liquid nitrogen before insertion into a precooled cryoholder (Gatan, Model 626,
Pleasanton, CA).
2.4
The Grid-Stripping Method
The so-called “grid-stripping” method was developed to study the lateral lipid
packing in intact SC from skin obtained from cosmetic surgery (ex vivo) or directly
from the flexor forearm of healthy volunteers (in vivo) in the TEM. The method
is based on the conventional tape-stripping technique. An advantage of this
method is that the plane of the lipid lamellae is perpendicular with respect to the
incident electron beam. In such an orientation the lateral packing can be studied
best because the different lattice planes are simultaneously exposed in a proper
way (see Fig. 5).
In this method, 400-mesh copper grids are plunged into a glue—chloroform
solution (40 mg of Avery T406 glue per milliliter of chloroform). After the
excess of chloroform has been blotted away with filter paper, the grids are air-
dried, leaving a sticky layer on the grid bars. These “glue grids” are used for
direct collection of the SC strips from either ex vivo or in vivo skin. Samples
prepared in this way will be referred to as “grid strips” (Fig. 6A). Conventional
tape strips are taken with cellotape to remove the unwanted layers before the
depth of interest in the SC. Thus, by varying the number of tape strips that are
removed before a grid strip is taken, it is possible to study the lipid layers of human
SC at various depths. Mostly, after removal of every two to four conventional
tape-strips, three glue grids are placed rough side down onto the tape-stripped
site of the skin. The corneocytes that adhere to the glue on the grid bars partly
float over the holes of a grid. These protrusions, which are covered with lipid
layers, can be studied in the TEM (Fig. 6B). Because a grid strip removes only a
small amount of SC, the next tape strip is used to remove the remnant of the
same layer of the SC, after which successive tape strips are used to reach deeper
layers of the SC. For ex vivo skin, approximately 17 tape strips will remove
most of the SC, whereas up to 30 tape strips are required for in vivo skin.
Cryo fixation of the lipids is done by plunging the grids into liquid nitrogen
cooled ethane either directly (in vivo skin at 32°C) or after equilibration to 32°C
(ex vivo skin) or room temperature. The SC grid strips are stored in liquid
nitrogen until use.
FIGURE 5 Schematical representation of the parallel and perpendicular orientation of

the SC lipid layers with respect to the incident electron beam. In the parallel
configuration, only one set of repetitive lattice planes (lateral packing) of a crystal can be
properly oriented at once, which results in unidirectional scattered ED patterns. As a
consequence, it cannot be determined whether 0.41 nm reflections are derived from the
hexagonal or orthorhombic lattice. When both the 0.37 and 0.41 nm reflections are
present, these must be derived from crystals of at least two different orientations. In the
perpendicular orientation, the various lattice planes give rise to reflections in the ED
pattern simultaneously. Based on the location of the reflections, it may become possible to
determine whether these belong to the hexagonal or orthorhombic lattice. (From Ref. 95.)
3
THE LIPID ORGANIZATION IN STRATUM CORNEUM
3.1
The Lamellar Phases Studied by Small-Angle X-Ray
Diffraction
Figure 7 shows small-angle X-ray diffraction (SAXD) curves of human SC
measured at room temperature and after recrystallization [31]. The curve
obtained at room temperature is characterized by a strong and a weak diffraction
peak. Both peaks consist of a main position and a shoulder on the right-hand side
(Fig. 7A). Since the peaks are limited in number, very broad, and partly
overlapping, additional information is required for proper interpretation. This is
obtained in X-ray experiments with SC for which the lipids are recrystallized
from 120°C to room temperature. The diffraction curves reveal the presence of a
series of sharp peaks, similar to those noticed in mouse SC [32, 33], (Fig. 7A).
Such a diffraction profile is characteristic for a lamellar phase. From the
positions of the peaks, the periodicity d of the lamellar phase can be calculated
according to Bragg’s law (see Sec. 2.1). In our work, such calculations revealed
that after recrystallization, the lipids in SC are organized in a lamellar phase with
FIGURE 6 (A) Schematic drawing of the tape- and grid-stripping method. Tape strips
can be used to remove SC layer by layer until the depth of interest has been reached.
Subsequently, a glue grid can be applied to remove some corneocytes, which are covered
with lipid lamellae. (B) Scanning electron micrograph of a grid strip. Corneocytes are
shown which adhere to the gridbars; one protrudes over a hole and can be studied with
TEM, and another is located on top of the gridbar. (Figure 6B is reprinted from Ref. 40.
Copyright is held by the Royal Microscopical Society.)
a periodicity of 13.4 nm. Comparison of the peak positions in the diffraction
patterns obtained before and after recrystallization revealed the presence of at
least two lamellar phases: one with a periodicity of approximately 6 µm (referred
to as the short-periodicity phase), and the other with a periodicity of approximately
13 nm (the long periodicity phase) [33].
Another major difference noted before and after the recrystallization of SC
lipids is the width of the diffraction peaks at half-maximum. In untreated SC the
width at half-maximum is very broad. This is because the limited space between
the corneocytes limits the number of lamellae between the corneocytes. Electron
microscopic observations indicated that the number of repeating units (i.e., the
broad-narrow-broad sequence) between the cells does not exceed 3 to 4 (broad-
narrow-broad sequences after RuO4 fixation) [7]. After recrystallization of the
lipids, however, the width at half-peak-maximum is much smaller. Recently we
used electron microscopy in combination with RuO4 post-flxation to visualize
human SC after heating and recrystallization of the lipids. The electron
micrographs revealed that during the heating and cooling process SC lipids
migrate to the surface of the SC and recrystallize in lamellae with a broad-
narrow-broad sequence [34]. On the skin surface the number of lamellae in the
stacks is not limited by the space between corneocytes, which is probably why a
series of sharp peaks is present in the diffraction pattern after recrystallization.
To obtain more detailed information on the SC lipid organization, changes in
diffraction pattern as a function of temperature also were investigated. These
FIGURE 7 SAXD and WAXD patterns obtained with human SC. In the SAXD curves
the intensity is plotted as a function of Q, the scattering vector, defined as 4 sin / , in
which is the scattering angle and A, is the wavelength of the X-rays. (A) The SAXD
curve of human SC at room temperature and after recrystallization from 120°C. After
recrystallization the peaks are located at equal distances, strongly indicating a lamellar
phase with a periodicity of 13.4nm. This phase is referred to as the long-periodicity phase
(LPP). Comparing this curve with the curve at room temperature revealed the presence of
the second lamellar phase in human SC with a periodicity of 6.4 nm, referred to as the short-
periodicity phase (SPP). 1, 2, …, 6 denote the firsts, second,…sixth-order peaks of the
pattern based on the LPP; I and II refer to the first and second order of the SPP.
(Reprinted by permission of Blackwell Science, Inc. from Ref. 31.) (B) The temperature-
induced changes in SAXD profiles of human SC. The heating rate was 2°C/min. Each
sequential curve has been monitored for one minute. The lamellar phases disappear
between 60 and 75°C. The first order diffraction peak is clearly depicted, and the asterisk
(*) denotes the peak attributed to the first order of the SPP and the second order peak of the
LPP. CHOL indicates the peaks attributed to phaseseparated crystalline CHOL. (From
Ref. 35.) (C) WAXD pattern of human SC oriented parallel to the primary X-ray beam.
The diffraction pattern is characterized by two rings indicating that the lipids are
organized in an orthorhombic lateral packing. The rings are stronger in equatorial position,
indicating that the lipids are oriented mainly perpendicularly to the SC surface.
Furthermore, a number of reflections can be attributed to phase-separated CHOL. The
position of the reflection in the pattern indicates that CHOL crystals have a preferred
orientation in a direction similar to that of the lipid lamellae. The strong broad reflections
at 0.46 and 0.92 nm can be attributed to soft keratin. (From Ref. 37.)
experiments revealed that lipid lamellae are still present until a temperature of
around 60°C, after which the lipid lamellae disappear within a temperature range
of approximately 10°C (see Fig. 7B) [35]. Since the hydration level in vivo can
vary quite substantially, the effect of hydration on the lipid organization in SC is
of interest. From these studies it is obvious that an increase in the SC water
content from 20 to 60% w/w does not lead to a change in the peak positions and
therefore does not lead to a swelling of the lipid lamellae in the SC (not shown).
3.2
Lateral Packing Studied by Wide-Angle X-Ray Diffraction
The WAXD pattern revealed two strong diffraction rings at approximately 0.41
and 0.37 nm, indicating that the lipids form an orthorhombic packing in SC [36, 37].
Whether a hexagonal or a fluid phase coexists in addition to this crystalline

packing cannot be concluded from this pattern, since the diffraction ring
attributed to hexagonal lateral packing is obscured at approximately 0.41 nm by
the strong diffraction peak attributed to the orthorhombic phase, (Fig. 7C). There
are some other interesting features present in the diffraction pattern of SC. The
presence of soft keratin in the corneocytes results in two broad rings at spacings
of approximately 0.45 and 0.95 nm. Since the diffraction ring that can be
attributed to a liquid phase is also expected to be present at a spacing of 0.45 nm,
it is impossible to determine the existence of a liquid lateral packing in intact SC
from the diffraction patterns of intact SC. However, the presence of a liquid
phase has been established by Fourier transform infrared spectroscopy (FTIR)
[38, 39] and more recently in lipid mixtures prepared from isolated ceramides
(see later). From the above-mentioned studies, we can conclude that the SC
lipids have an exceptional organization, not observed in other biological
membranes. Most probably this is caused by the presence of the long-chain
ceramides and the absence of phospholipids. The head groups of the ceramides
contain several hydroxyl groups that can form lateral hydrogen bonds with
adjacent lipids. As a result, the lipids in the lamellae are very tightly packed.
Furthermore the lipids are very hydrophobic, and the lamellae do not swell upon
hydration.
3.3
Electron Diffraction of Healthy Human Skin In Vivo and Ex
Vivo
In Section 2.2 it is explained that ED provides information on the lateral packing
of SC lipids that is supplementary to that obtainable from WAXD. However, the
lipids should be properly oriented (Fig. 5), which can be achieved using the grid-
stripping method described in Section 2.4 [40]. This was demonstrated in SC
samples upon tilting the grid strips in the TEM (Fig. 8). At an angle of 10° with
respect to the incident electron beam, reflections already begin to disappear.
Tilting angles of 30° or more result in unidirectional scattered ED patterns,
provided a set of repetitive lattice planes is present perpendicular to the tilt axis
[41].
Both in vivo and ex vivo human skin were studied in relation to depth in the
SC. Some characteristic ED patterns are shown in Figure 9. No differences were
observed between ED patterns from in vivo and ex vivo SC. The diffraction
patterns mostly consisted of concentric rings as in WAXD, or of opposite arcs or
spots at both 0.41 and 0.37 nm, especially when smaller areas were selected for
ED. These latter ED patterns are important to distinguish whether only
orthorhombic packing is present or hexagonal packing as well. Furthermore,
faint reflections at 0.22 and 0.25 nm were recorded occasionally. These
reflections can be attributed to other sets of lattice planes of the same crystal
FIGURE 8 Tilting series obtained at the same area shows that reflections begin to
disappear already at small tilting angles. (a-d) ED patterns obtained at tilting angles 0°,
10°, 30°, and 0°, respectively. In (a) the tilt axis is indicated by a dashed line. In (d) the
arrow indicates a 0.41 nm reflection and the arrowhead marks a 0.37 nm reflection, which
became restored after tilting back to the original position. (Reprinted by permission of
Blackwell Science, Inc. from Ref. 41.)
indicated in Figure 3C. The spacings are in agreement with calculated values and
with the spacings established by WAXD.
To get an overview of the data, the ED patterns were classified into four
categories: orthorhombic (ort), orthorhombic in which hexagonal cannot be
excluded (ort*), hexagonal (hex), and patterns that are probably hexagonal
(hex*). The relative distribution of the ED patterns in these categories was
similar for ex vivo and in vivo skin (not shown). The data in Figure 10 are
plotted as a function of depth in SC and show that the percentage of ED
patterns attributed to the hexagonal lattice increased at 32°C compared with room
temperature. Throughout the SC the orthorhombic packing prevailed: in the
upper part of the SC, however, the hexagonal lattice could occasionally be
detected as well. From this it was concluded that ED patterns attributed to
hexagonal packing were recorded mainly in the superficial layers of the SC at
(figure legend on p. 128)

FIGURE 9 Characteristic ED patterns of both in vivo and ex vivo human SC. (a) An
orthorhombic sublattice that can be found throughout the SC; arrow indicates 0.41 nm
reflections; arrowhead marks 0.37 nm reflections. (b-c) Respectively, two and three
orientations of orthorhombic crystals rotated approximately 60° with respect to each other.
As a consequence, the 0.41 nm reflections are obtained from two differently oriented
crystals (41). (c) Higher order reflections are indicated by the arrowhead (0.24 nm) at an
interplanar angle of 90° with respect to the 0.37 nm reflection, and the arrow (0.22 nm).
(a-c) Also shown in a schematic representation of the corresponding orientations of,
respectively, one, two, and three orientations of the orthorhombic lattice. The double-
sided arrows indicate the direction of the 0.37 nm reflection. (d) Two rings at 0.37 and 0.
41 nm in which the hexagonal lattice cannot be excluded, as in WAXD patterns. (e) A
hexagonal lattice that is mainly found in the outer layers of the SC. (Adapted from Ref. 95.)
room temperature and at 32°C. In vivo, however, one would expect to observe
the hexagonal lattice more frequently in the lower part of the SC, where the
temperature approaches 37°C, but this was not the case. Therefore, there should
be another explanation for this phenomenon, which might be the presence of
sebaceous lipids that are secreted on the surface of the skin and partly penetrate
the intercellular matrix of the SC [42, 43]. Such sebaceous lipids may form a
FIGURE 10 Distribution of hexagonal and orthorhombic lattices in relation to depth in ex

vivo human SC equilibrated to room temperature or 32°C. The number of recordings (N)
shown on the x axis is set at 100% for each depth indicated by grid-strip numbers 3, 6, 10,
and 17. The percentage of ED patterns per category is shown along the y axis (ort,
orthorhombic; ort*, orthorhombic, however, the hexagonal lattice cannot be excluded;
hex, hexagonal; hex*, probably hexagonal). (Reprinted by permission of Blackwell
Science, Inc., from Ref. 41.)
hexagonal packing or create a phase transition within the endogenous lipids from
orthorhombic to hexagonal. FTIR studies by Bommannan et al. [42] showed a
fluidization of the upper SC lipids, while Golden et al. [44] have shown that the
unsaturated fatty acids may function as penetration enhancers. Because sebum
mainly consists of glycerides, squalene, and wax/sterol esters as well as (short-
chain) free fatty acids, it is very likely that these substances may alter the
endogenous lipid structure by increasing alkyl chain mobility.
The phase transition from orthorhombic to hexagonal occurs between 35 and
40°C. This phase transition could also be studied online in the TEM. It was
noticed that the 0.37 nm reflection moves gradually towards the 0.41 nm
reflection, indicating that the packing density of the structure is gradually
increased. Above 40°C only the hexagonal packing was detected, and above 80°
C the lipids formed a liquid phase (Fig. 11).
As with WAXD, the possible presence of a liquid phase around 0.46 nm at
physiological temperature is difficult to establish by means of ED, because the
-keratin inside the corneocytes produces a broad reflection with the same
spacing. Yet, sometimes a broad band with a sharp edge can be distinguished,
which may be attributed to lipids in the liquid lamellar phase (see also Sec. 3.4).
Furthermore, a reflection between 0.48 and 0.50 nm that can be attributed to
crystalline cholesterol was occasionally observed. Reflections in this area have
also been observed in WAXD studies [37, 45]. Upon treatment of the SC sample
with a mixture of chloroform and methanol, this reflection was no longer visible,
indicating that it was of lipid origin indeed [40].
FIGURE 11 Temperature series showing the lipid phase transitions obtained by

increasing the temperature of the sample during examination. (a-d) Obtained at,
respectively, RT, 35°C, 60°C, and 90°C. (a) ED pattern in which reflections of the
hexagonal lattice may be obscured by orthorhombic ones. All reflections in the inner ring
at 0.41 nm, however, correspond to 0.37 nm reflections in the outer ring (one orientation
is indicated). (b) The original 0.37 nm reflection moves gradually toward the 0.41 nm
reflection, indicated by the arrowhead. (c) Hexagonal lipid packing in which the
interplanar angle of the reflections is 60°. A broad band with a sharp edge that might be
indicative of lipids in the fluid phase can already be observed. (d) All lipids are in a fluid
phase, characterized by a broad band around 0.46 nm. (Reprinted by permission of
Blackwell Science, Inc., from Ref. 41.)
Another striking observation was that the ED patterns frequently displayed

reflections forming three pairs of double arcs at 0.41 and 0.37 nm. These
reflections can be assigned to the orthorhombic lattice only by rotating three
successive orthorhombic crystals over an angle of 60° relative to each other
(Fig. 9a-c). These three orientations may be present within the same lamellae or
superimposed on top of each other. The latter possibility could be in good
agreement with a recently proposed molecular model for long-lamellar
periodicity [19, 46], and with the broad-narrow-broad (Landmann unit) sequence
visible in SC sections stained with ruthenium tetroxide [3], which suggest that
these lamellae consist of three lipid layers. The alignment of the lipid layers is
probably of importance for a proper barrier function, because mismatches
between crystallites, occurring when the lateral or lamellar organization is not
maintained, are sites at which permeability for compounds may increase [47].
3.4
Diseased Skin
Impairment of human SC barrier function can lead to increased transepidermal
water loss (TEWL) and increased susceptibility to intoxication by penetration of
harmful agents. This has been observed in various skin diseases [48–50]. In certain
skin diseases in which the barrier function is affected, aberrations in lipid
composition have been described. Two examples of such diseases are atopic
dermatitis (AD) [51–53] and lamellar ichthyosis (LI) [54]. It has been shown that
patients suffering from AD have a reduced ceramide content, especially CER 1,
in the SC, whereas in the SC of LI patients, the amount of long-chain free fatty
acids is decreased and the ceramide profile is altered. Since ceramides and free
fatty acids are essential for a proper barrier function, we used ED to investigate
whether the changes in the composition of these lipids would be reflected in the
lipid organization in SC of AD and LI patients.
The lateral lipid organization in SC of the flexor forearm of three healthy
volunteers was compared with that of three AD patients and three LI patients.
Grid strips were collected at depths 2 and 8 and immediately frozen in ethane
cooled with liquid nitrogen (see Sec. 2.4). Figure 12 shows some characteristic
ED patterns that have been recorded in the SC of healthy, AD, and LI skin. The
recorded ED patterns were classified into five categories: ort, orthorhombic; ort*,
orthorhombic, although the hexagonal cannot be excluded; hex, hexagonal; hex*,
probably hexagonal, although faint reflections of the orthorhombic lattice may be
present; and ort+hex, both orthorhombic and hexagonal present in the same area
selected for ED. We added the ort+hex category because combinations of them
were observed regularly in diseased SC. In this category, more crystals of
different types are present in a relatively small area, which indicates that crystal
mis-match occurs. Therefore, we decided not to assign these patterns to one of the
other four categories. The distribution profiles of the ED patterns that have been
collected from the SC of the healthy volunteers and the AD and LI patients at
two depths are depicted in Figure 13.
As already described in Section 3.3, orthorhombic packing predominates in SC
of healthy skin at both depths. Hexagonal packing is observed more frequently at
depth 2 than at depth 8. Although statistical analysis could not establish a
significant difference between the patterns at strips 2 and 8, a similar trend
related to a decrease of the hexagonal lattice with depth was observed, as in the
ex vivo samples shown in Figure 10.
FIGURE 12 Representative ED patterns recorded in diseased skin. (a) Orthorhombic

pattern recorded on a grid strip of an AD patient. The reflections appear as small spots.
(b) Hexagonal and orthorhombic lattices in the same area selected for diffraction (ort +
hex). This pattern was observed occasionally in SC from LI patients. (c) Hexagonal
pattern, frequently observed in samples from LI patients (hex). The pattern shown in (c)
could also be observed in samples from AD patients. (Reprinted by permission of
Blackwell Science, Inc., from Ref. 64.)
FIGURE 13 Relative distribution of the ED patterns into the five categories from
controls, AD, and LI patients. The graph summarizes the ED patterns recorded in samples
from healthy volunteers (C) and from AD and Ll patients in relation to treatment and
depth (strip numbers 2 and 8). The presence of hex is significantly increased in the SC of
AD patients and predominates in the SC of Ll patients;. N is the number of recordings.
(Adapted from Ref. 64.)
The lateral lipid organization of AD skin showed an aberrant distribution

profile of ED patterns in comparison to the healthy controls. The frequency of
hexagonal packing was significantly increased at both depths. Yet, orthorhombic
packing was regularly observed, although the reflections more often appeared as
small spots, which indicate the presence of differently oriented crystals.
Yamamoto et al. [52] showed that instead of linoleate (C18:2), oleate (C18:1) is
esterified to CER 1 and that the fraction of fatty acids esterifled to CER is
slightly more mono unsaturated. The mono unsaturation of the esterified fatty
acids (kink in the acyl chain) may prevent the lipids from organizing in a
crystalline lattice. The decrease in CER content has been associated with the dry
appearance of AD skin, as well as the reduced amount of sebaceous lipids [55]
due to reduced activity of sebaceous gland secretion [56]. In 1999 Bleck et al.
[57] showed that in the upper part of the SC the relative amount of FFA is
decreased. However, it remained uncertain whether this is caused by a reduced
amount of FFA from sebum or by an increased loss of FFA. The origin of the
FFA fraction is crucial because in lipid mixtures containing pig CER (or human
CER), it has been observed that in the absence of long-chain FFA a hexagonal
packing is predominantly present [19, 58–30]; see Section 4. Therefore,
alterations in hydrocarbon chain saturation, a decreased amount of CER 1, and
possibly long-chain FFA in SC of AD patients may account for a reduction of the
orthorhombic lattice.
The changes in the distribution profile of the ED patterns collected from the
skin of LI patients were even more pronounced. In these patients, the hexagonal
lattice in SC clearly predominated at both depths. The orthorhombic ED patterns
differed from those of control samples, comprising mainly small faint spots next
to the hexagonal lattice; a fluid phase was clearly observed at physiological
temperature. Lavrijsen et al. [54] showed in studies on the SC lipid composition
of the same LI patients that the FFA/CER as well as the FFA/CHOL ratios were
lowered, while the CHOL/CER ratio remained similar to that in normal skin.
Besides the reduced FFA content, the composition of the CER fraction differed
from that of control SC. In 1999 it was demonstrated that a change in the CER
composition (except for a reduction in CER 1) does not alter the phase behavior
in equimolar mixtures of CHOL and porcine CER [61]; see also Section 4.
Therefore, reduced FFA levels form the most likely explanation for the
predominant hexagonal packing in SC of LI patients. However, since a reduction
in the chain length of either CER or FFA might also occur, the possibility that
this may play a role as well cannot be excluded.
The changes in the lateral lipid organization observed in the SC of both groups
of patients may account for the aberrations in SC barrier function. Several
studies of lipid phase behavior in both phospholipid membranes [62] and SC
[63] have shown that coexisting phases (e.g., during phase transitions) lead to
increased permeability of certain compounds. The reason for this may be an
increase of alkyl chain mobility, as a result of which compounds can cross lipid
layers more readily. Furthermore, lipid layers may show leakage between grain
boundaries, which arise at sites at which two or more crystals of differing natures
fail to form a continuous layer [47]. As a result, penetration of compounds may
be enhanced. Such an effect is very likely to occur in the SC of both patient
groups as evidenced by our finding that the number of crystal orientations in a
certain area selected for diffraction was increased. Furthermore, hexagonal and
orthorhombic lattices could be observed in the same ED patterns.
Additionally, other explanations for a defective barrier remain possible. By

using freeze fracture electron microscopy (FFEM) we have shown that
aberrations are present with respect to the lamellar organization; moreover the
morphology of corneodesmosomes was altered in both patient groups [64].
Furthermore, Fartasch et al. [65] demonstrated that the lamellar extrusion
process in AD skin is impaired, whereas this process appears normal in LI skin
despite the apparent incompleteness of the reorganization of the lipid stacks into
lamellar sheets [66, 67]. In an earlier study by Lavrijsen et al. [54], a decrease in
the lamellar spacings in LI skin was observed as measured with SAXD, which
indicated major changes in the lamellar phase behavior in comparison to normal
skin. These investigators suggested that either changes in CER composition or a
reduction in CER or FFA chain length might explain the decrease in lamellar
periodicity. Other studies on LI skin reported changes in the corneocyte envelope
[68–70] as well as defects in the gene coding for transglutaminase, an enzyme
that participates in the formation of the cornified envelope by catalyzing the
cross-linking of precursor proteins such as involucrin [71]. These findings may
also help to explain the impaired SC barrier function. Thus, it seems feasible to
suggest that SC barrier function depends on both lipid organization and protein
structures.
4
THE LIPID PHASE BEHAVIOR OF LIPID MIXTURES
PREPARED WITH ISOLATED CER
It is very essential to have detailed information on the lipid organization in intact
SC. To understand penetration pathways in the SC and the deviation in lipid
phase behavior in diseased and dry skin (see Sec. 3.4), however, it is also crucial
to obtain information about the role of the various lipid classes in SC lipid phase
behavior. Thus we initiated a research program to study the phase behavior of lipid
mixtures prepared from isolated pig ceramides (pigCER). First the phase
behavior of mixtures of CHOL and pigCER was studied by varying the CHOL:
pigCER molar ratio systematically. An equimolar mixture of CHOL and pigCER
was prepared at a pH of 5 and examined [58] by means of SAXD and WAXD. In
the SAXD pattern the presence of a large number of sharp peaks was noticed
(Fig. 14). Peaks I and II were assigned to a lamellar phase with a periodicity of
5.2 nm, and peaks 1, 2, 3, 5, and 7 to a lamellar phase with a repeat distance of
12.2 nm. Furthermore, two additional peaks at 3.35 nm and 1.68 nm were
detected. These can be attributed to CHOL, which was not dissolved in the
lamellar phases, but phase-separated and formed crystalline domains of CHOL.
Reducing the CHOL: pigCER molar ratio to 0.4 did not change the phase
behavior, except that a smaller amount of CHOL phase-separated in crystalline
domains. Only if the CHOL: pigCER molar ratio reduced to 0.2 or increased to 2
did a smaller fraction of lipids form the 12.2 nm phase.
FIGURE 14 The phase behavior of equimolar CHOL: CER and the CHOL-CER: FFA
mixture prepared at pH 5. The arabic numbers indicate the diffraction orders of the long-
periodicity phase (repeat distance between 12 and 13 nm). The roman numbers indicate
the diffraction orders of the short periodicity phase (repeat distance between 5.3 and 5.5
nm). (From Ref. 93.)
As can be inferred from Figure 15 A, in which the wide-angle pattern is

plotted, a single peak at 0.405 nm dominates the diffraction profile. This strongly
indicates that the lipids in the lipid lamellae mainly form a hexagonal lateral
packing. Addition to the lipid mixtures of the third major component, long-chain
FFA, did not change the long-range ordering dramatically. Again, two lamellar
phases are present with repeat distances of approximately 5.4 and 12.8 nm
(Fig. 14), similar to the result observed in the mixtures of CHOL and pigCER.
In contrast to the lamellar phase behavior, addition of free fatty acids induced
a phase change in the lateral packing from hexagonal to orthorhombic packing
[59], as demonstrated by the sharp peaks at 0.375 and 0.415 nm (Fig. 15B). From
this observation it is obvious that the free fatty acids increase the lipid packing
density, which may be very important for the skin barrier function. An increase
in lipid density is expected to reduce permeability across the membrane.
The lipid phase behavior of mixtures of CHOL, pigCER, and FFA has also
been studied online as function of temperature. The heating rate was 2°C/min,
and a diffraction curve was recorded each minute. In this way the lipid phase
changes could be studied online as function of temperature (Fig. 16A). The
sequential SAXD curves obtained between 25 and 95 °C of the equimolar CHOL:
CER: FFA mixture remained unchanged [35] until a temperature of
approximately 35°C was reached. Then the formation of a new phase was
initiated, which became very dominant at elevated temperatures. Compared with
intact SC, this phase is formed at higher temperatures by only a small fraction of
lipids in intact SC. This indicates that the CHOL:-pigCER: FFA mixture mimics
SC lipid phase behavior at room temperature and at 32°C, the temperature of the
skin surface.
At elevated temperatures, however, important differences exist between the
lipid mixtures in intact SC. Besides pigCER, CHOL, and FFA, it has often been
argued that cholesterol sulfate, although present in small amounts (typically 2–5
% w/w of the lipids) in SC, plays a very important role in the inhibition of
proteases. In the superficial SC layers, cholesterol sulfate is metabolized to
cholesterol by cholesterol sulfatase. This reduction of the cholesterol sulfate
level increases the activity of proteases and promotes the degradation of the
desmosomes [72].
Since cholesterol sulfate plays an important role in the desquamation process,
the effect of cholesterol sulfate in the lipid phase behavior was also studied.
Addition of only 2% m/m cholesterol sulfate to an equimolar CHOL: pigCER:
FFA mixture resulted in the reduction of the fraction of CHOL that formed
separate crystalline domains. Furthermore, a fluid phase was clearly present in the
CHOL: pigCER: FFA: cholesterol sulfate mixture (Fig. 15C). However, at room
temperature the presence of the lamellar phases was not affected (not shown).
The lipid phase behavior has also been studied as a function of temperature. In
the presence of only 2% m/m cholesterol sulfate, the formation of the 4.3 nm
phase is shifted to higher temperatures [35], mimicking the lipid phase behavior
of intact SC also at elevated temperatures (Fig. 16B). This observation suggests
that cholesterol sulfate stabilizes the lipid lamellar phases formed at room
temperature, perhaps as a result of electrostatic interactions induced by the
presence of the negatively charged sulfate group. A stabilization of the lamellar
phases after the introduction of cholesterol sulfate into the mixture has been
observed for mixtures containing sphingomyelin and phosphatidylcholine, as
well [73, 74]. From the results it is clear that cholesterol sulfate is required for
proper lipid phase behavior over a wide temperature range. When these findings
are extrapolated to the in vivo situation, it seems that cholesterol sulfate is
required to dissolve CHOL in the lamellar phases and to stabilize SC lipid
organization. Therefore, a drop in cholesterol sulfate content in the superficial
layers of the SC is expected to destabilize the lipid lamellar phases and to
increase sterol sulfatase activity. Both events will facilitate desquamation.
FIGURE 15 (A) One-dimensional WAXD pattern of the equimolar CHOLpigCER

mixture plotted as function of Q (see Fig. 4). A broad reflection at 0.415 nm spacing is
attributed to the presence of the hexagonal lattice. A large number of sharp reflections is
based on phase-separated CHOL. (B) One-dimensional WAXD pattern of the equimolar
CHOL: pigCER: FFA mixture. Two strong sharp reflections indicate the presence of an
orthorhombic lateral packing. (C) The one-dimensional WAXD pattern of the 1:1:1:0.06
CHOL: pigCER: FFA: cholesterol sulfate mixture. Two strong reflections are attributed to
the orthorhombic lateral packing. The broad reflection at 0.46 nm indicates the presence of
a liquid phase. Note that in the presence of cholesterol sulfate, the reflections attributed to
CHOL disappeared. (Reprinted by permission of Blackwell Science, Inc., from Ref. 59.)
The phase behavior of mixtures prepared from human ceramides (HCER) has
been studied, as well. These studies revealed that in mixtures prepared of HCER
with either CHOL or CHOL and FFA, that the long-periodicity phase dominates
in CHOL: HCER mixtures. Addition of FFA promotes the formation of the short-
periodicity phase and induces a transition from a hexagonal sublattice to an
orthorhombic one. Furthermore, the presence of free fatty acids promotes the
formation of a liquid lateral packing, and cholesterol sulfate reduces the amount
of CHOL that phase-separates in crystalline domains. Finally, in the absence of
HCER 1 the formation of the long-periodicity phase was strongly reduced.
From these observations it was concluded that the phase behavior of mixtures
prepared with HCER is similar to that of mixtures prepared with pigCER. Only
two differences were observed. In HCER mixtures the FFA slightly reduced the
presence of the long-periodicity phase and increased the formation of the liquid
FIGURE 16 (A) The temperature-induced changes in SAXD pattern of the equimolar

CHOL: pigCER: FFA mixture. Note the formation of a new peak at 4.3 nm (close to the
third order of the long-periodicity phase) at around 35°C. The lamellar phases disappear
between 60 and 80°C, except for the 4.3 nm phase, which is still present at 90°C. CHOL
indicates the peaks attributed to phase separated crystalline CHOL. (B) The temperature-
induced changes in SAXD pattern of the CHOL: pigCER: FFA: cholesterol sulfate (molar
ratlo:1:1:1:0.06) mixture. The formation of a new peak takes place at much higher
temperatures, and the intensity of this peak decreased strongly in comparison to that
shown in (A). (From Ref. 35.)
lateral packing. These effects were not observed in mixtures prepared from
pigCER.
5
THE “SANDWICH MODEL” AS A HYPOTHESIS FOR
LIPID ORGANIZATION
Having obtained information on lipid phase behavior in SC and in lipid mixtures,
the next step is to construct the molecular arrangement in the lipid lamellae based
on the experimental data. Since the long-periodicity phase is present in all
species examined until now and is characteristic of the lipid organization in SC,
it has been suggested that the presence of this phase plays an especially
important role in skin barrier function. Therefore,we focus mainly on the
molecular arrangement of this phase. Figure 17 gives the molecular model based
on the composition of HCER. However, a similar molecular model can be
derived from mixtures prepared with pigCER. As can be inferred from
Figure 17, we propose a trilayer arrangement for the long-periodicity phase. A
trilayer arrangement has also been suggested by Swartzendruber et al. [46] and
by Kuempel et al. [75]. The trilayer arrangement is based on the broad-narrow-
broad sequence of electron lucent layers seen in SC fixed in ruthenium tetroxide

and the broad-narrow-broad sequence of low electron density regions within the
repeating structure of the long-periodicity phase. This sequence is based on
electron density calculations from the intensities of the diffraction peaks
attributed to the 12.2 nm phase in CHOL: CER mixtures (see Sec. 4). The
molecular model contains only CER and CHOL, since these two classes of
lipids are important for the formation of the long-periodicity phase (Sec. 4).
Incorporation of FFA into the molecular model would not change its basic
features. The model and its experimen-tal evidence is explained in more detail in
the seven points that follow.
1. The linoleate moieties of HCER 1 and of HCER 4 are located in the narrow
central layer of the long-periodicity phase and link the trilayers together (in
the case of pigCER, this is only pigCER 1) . This is based on the important
role of CER 1 in proper lipid phase behavior. Earlier studies (Sec. 4) showed
that in equimolar CHOL: pigCER and CHOL: pigCER: FFA mixtures
varying in pigCER composition, the lipids were organized into long- and
shortperiodicity phases, similar to what is observed in intact SC [61]. With
equimolar CHOL: pigCER and CHOL: HCER mixtures in the absence of
CER 1, however, the long-periodicity phase was only weakly present [19],
indicating that the CER 1 plays a crucial role in the formation of the long-
periodicity phase.
2. The liquid sublattice is located in the central lipid layer of this phase. In the
intercellular domain of the SC, the orthorhombic phase coexists with a liquid
phase. This has been established by FTIR spec troscopy [38, 39] and WAXD
[59, 76] in human SC and in mixtures prepared with native CER,
respectively. In the sandwich model it is proposed that the liquid phase is
located in the central narrow layer, in which unsaturated linoleate linked to
-hydroxy acid of CER 1 (and HCER 4 for human SC) is present. The
relationship between the linoleate/oleate moiety and the presence of a liquid
phase was confirmed by a recent study in which natural HCER 1 was
replaced by either synthetic CER 1-linoleate, by CER 1-oleate, or by CER 1-
stearate. No liquid phase could be detected when natural HCER 1 was
substituted for CER 1-stearate, while substitution of HCER 1 by either CER
1-oleate or CER 1-linoleate clearly revealed the presence of the liquid
phase, indicating the major influence of the unsaturated acyl chains. In the
same study it was observed that for the formation of the long-periodicity
phase, a certain (optimal) fraction of lipids must form a liquid phase [77].
3. In the sublattice adjacent to the central layer, the presence of less mobile
long saturated hydrocarbon chains causes a gradual change in lipid
mobility. This results in densely packed lipid layers on both sides of the
central layer and avoids the formation of new interfaces, which would be
energetically unfavorable. The presence of crystalline orthorhombic packing
FIGURE 17 The molecular arrangement of the long-periodicity phase in HCER: CHOL

mixtures. Following this model the repeating unit in the structure consists of three lipid
layers. From this model it is obvious that HCER 1 and HCER 4 are important for the
formation of this phase. Furthermore, the unsaturated linoleic moieties of HCER 1 and
HCER 4 are located in the central narrow layer of this model. Based on the molecular
model, we proposed the “sandwich model” [93]. The characteristics are as follows: (1) The
liquid sublattice is located in the central lipid layer of this phase. In this layer mainly
unsaturated linoleic acid and cholesterol are present. (2) In the sublattice adjacent to the
central layer, there is a gradual change in lipid mobility owing to the presence of less mobile
long saturated hydrocarbon chains (see Fig. 1). This gradual change to crystalline packed
lipid layers on both sides of the central layer avoids the formation of new interfaces. (3) Only
a small fraction of lipids forms a fluid phase in the SC. Therefore, one can assume that
this central lipid layer is not a continuous fluid phase. (4) Lamellae are mainly oriented
parallel to the surface of the corneocytes. Since the liquid phase is the most permeable
phase, it is assumed that the penetration pathway of small molecules parallel to the
lamellae (x-y direction, see Fig. 2) is mainly through the liquid channels or domains in the
central narrow layer if the LPP (large arrows). This will facilitate communication between
the desmosomes. By passing the SC lipid regions in the direction perpendicular to the
basal plane (z-direction; see Fig. 2), the crystalline domains can be circumvented (arrow
heads) by permeation (a) along the boundary between two crystalline domains within a
lamellar phase, (b) between the boundaries of two adjacent lamellar sheets, and (c)
between the lamellar sheets and desmosomes. (Reprinted by permission of Blackwell
Science, Inc., from Ref. 59.)
and a liquid phase has been demonstrated by WAXD, FTIR spectroscopy,

and ED (see earlier).
4. Only a small fraction of lipids forms a fluid phase in the SC. Therefore, one
can assume that this central lipid layer is not a continuous fluid phase, but
crystalline domains coexist. In this respect cholesterol may play a role as a
line active substance between crystalline and fluid domains, thereby
avoiding phase boundaries [78].
5. A trilayer arrangement might explain the two and three orientations of the
orthorhombic lattice often encountered in SC studied by ED. The lattice
planes of these three orientations are rotated approximately 60°. The
presence of an intermediate fluid phase may be required to align the
differently oriented orthorhombic lattices in the outer layers with domains of
the third orientation in the intermediate layer.
6. Lamellae are mainly oriented parallel to the surface of the corneocytes, as
demonstrated by electron microscopy studies. In passing the SC lipid
regions in the direction perpendicular to the basal plane, substances must
pass the crystalline lipid lamellar region and can only partly diffuse through
the less densely packed lipid regions parallel to the basal planes. In this way,
a tortuous route is created, providing an excellent barrier.
7. The presence of fluid regions may facilitate the deformation of the lipid
lamellae in the SC, especially in the case of shear stresses perpendicular to
the stacking direction.
As far as the short-periodicity phase is concerned, a “classical” bilayer

arrangement is the most likely arrangement. In this arrangement the hydrocarbon
chains deeply interdigitate in the lipid bilayer. The suggestion that ceramides can
be arranged in bilayers, which is of particular interest for biological membranes
was made by Dahlén and Pascher as long ago as 1979 [79].
In a much more recent paper, another model has been proposed for the SC
lipid organization, the single gel phase model, according to which the
intercellular lipids within the SC exist as “a single and coherent gel phase” and
“this structure has virtually no boundaries” [80], This gel phase is defined as a
“crystalline lamellar lipid structure that usually has a hexagonal hydrocarbon chain
packing.” Although this model is similar to our sandwich model with respect to
the absence of grain boundaries, the single gel phase model proposes in the
lower layers of the SC either the hexagonal phase or coexistence of the
hexagonal and orthorhombic phases such that the orthorhombic phase is located
close to the head group region and changes gradually into a hexagonal phase in
the central region of the lamellae. If this is the case, the orthorhombic and
hexagonal phases should always be present simultaneously. However, as
explained earlier (Sec. 3.3), no evidence has been found for the presence of a
hexagonal phase in the lower SC regions. In addition, the orthorhombic phase
was frequently observed without the presence of a hexagonal phase. Therefore,
the “single gel phase model” is not confirmed by experimental data. Finally, in
discussing “the single gel phase model” no attention has been paid to the role of
individual lipids in the lipid organization, including the crucial role of CER 1 in
the formation of the 13 µm lamellar phase and its influence on the phase
behavior, and the presence (or absence) of long-chain free fatty acids, which
cause the formation of the orthorhombic packing in vitro as well as in vivo, as
shown in diseased skin.
6
DISCUSSION
The results discussed thus far clearly demonstrate how X-ray diffraction and ED
contribute to an elucidation of the SC lipid organization, which provides insight
into the barrier function of the skin. Both in vivo and ex vivo SC samples have
been studied, as well as SC lipid models containing either pigCER or HCER.
With respect to the study of the lateral lipid packing, WAXD and ED are
complementary techniques. We have shown that when ED is used, the presence
of the hexagonal lattice can be determined even though the ED pattern displays
orthorhombic reflections, as long as the reflections are separated in arcs or spots.
This requires the selection of areas that are small enough and contain few
different lattice orientations, which may be difficult in some samples. The small
sample size offers the possibility of collecting in vivo samples (Sec. 2.4) to
generate diffraction patterns, which even allows the localization of the various
phases as a function of depth in SC. Generating an overall impression of the
lateral lipid organization with ED is time-consuming, however, because ED
provides local structural information of small sample areas. In contrast to
ED,WAXD requires larger samples, which generate an integral image of the lipid
organization in a sample. This allows us to establish the relative amount of
different lipid phases when reflection intensities are measured. However, these
phases cannot be localized. The requirement for larger samples makes sample
preparation from in vivo human skin overly invasive for volunteers, and
therefore ex vivo skin is generally used in WAXD studies.
It is more difficult to establish by means of WAXD the occurrence of the
hexagonal lattice in the presence of reflections from the orthorhombic lattice, and
less detailed information on crystal orientation is obtained in perpendicularly
oriented samples owing to the formation of ring patterns (unless the crystals are
very large or have only few orientations, e.g., cholesterol).
The organization of phospholipids has been studied extensively for a long
time. For biological membranes, the lipid bilayer has long been viewed as a fluid,
homogeneous solvent for (trans) membrane proteins [81]. However, this model
is being revised, and the formation of laterally separated domains (also known as
rafts) [82] is now recognized as an important driving force in the regulation of
membrane protein activity and the arrangement of cell surface receptors [83–87].
These heterogeneous lipid distributions may be due to phase-separated, or
inhomogeneously mixed types of phospholipids, or to superlattice arrangements
of, say, cholesterol [86, 87]. Similarly, because of its heterogeneity, the SC lipid
organization is no longer viewed as a homogeneous barrier-forming lipid matrix.
Although studies on mixtures prepared with phospholipids generated much

information, one should be very cautious in applying these results to mixtures
prepared with ceramides, which differ in many aspects from phospholipids (e.g.,
by their small head groups, absence of charge, and long and saturated acyl
chains).
It is clear from our studies on human SC, and from other studies using FTIR,
that lipid polymorphism occurs in the intercellular domains. Unlike the case in
phospholipid membranes, orthorhombic packing predominates, but the presence
of a liquid phase and hexagonal packing has also clearly been established. By
using ED, it was possible to locate the hexagonal packing in the outer layers of
the SC.
In case of diseased human skin, a marked increase of hexagonal packing was
observed. In hexagonal packing, the lipid density decreases and the rotational
mobility of the hydrocarbon chains increases compared with orthorhombic
packing. Several studies showed a decreased barrier function in these types of
skin, and therefore we hypothesize that hexagonal packing is not sufficient to
establish a proper barrier and that the formation of an orthorhombic packing is
required to a certain extent.
Changes in lipid composition apparently induce the formation of hexagonal
packing, as a result of which the permeability of the SC increases (Fig. 18A).
This is in agreement with studies in murine SC, in which increased permeation
below 55°C has been associated with an increase in free volume (decreased
packing density) resulting from the phase transition from orthorhombic to
hexagonal [88]. Furthermore, in the SC of LI patients, the occasionally observed
orthorhombic packing was present only locally, as separate crystals. In AD
patients, ED patterns have been recorded that were also indicative of the
presence of differently oriented orthorhombic lattices. Therefore, the presence of
grain boundaries may play a role in the impairment of barrier function, as well
(Fig. 18B).
The hypothesis that the hexagonal packing may not function as an optimal
lipid organization with respect to the SC barrier is very interest ing with regard to
the results obtained with sebaceous lipids. It has been shown by others that
sebum partly penetrates the superficial layers in the SC. Using SC lipid models,
we showed that the addition of sebaceous lipids induced the transition from an
orthorhombic to a hexagonal packing (data not shown) [89]. In the outermost
layers of the SC, the barrier function may thus be reduced as a result of the
presence of a hexagonal lipid organization, owing to mixing with sebaceous
lipids. Indeed, several studies have suggested that the SC barrier is mainly
located in the inner part of the SC, the stratum compactum [90–92].
Rationalizations for this idea include not only the lipid organization, but also
such other features as the degradation of corneodesmosomes and changes in the
corneocyte envelope.
FIGURE 18 Models for the lipid organization in SC, which may explain the reduced
barrier function of diseased skin. (A) Phase transition from the orthorhombic to the
hexagonal lattice, resulting in decreased packing density and increased hydrocarbon chain
mobility, which causes increased permeability. (B) Grain boundaries arise between phase-
separated lipid domains, or between differently oriented crystals of the same type. As a
result of these mismatches, fluctuations in packing density occur and permeability may be
increased.
7
CONCLUSIONS
The presence of a liquid phase has now been established by using FTIR and
WAXD in intact SC as well as in lipid models. The liquid phase has also been
observed by means of ED in diseased skin (Sec. 3.4) and in ex vivo SC as well as
SC lipid models treated with enhancers [64, 89]. From the results of WAXD
studies on SC lipid models, it can be concluded that the fluid phase is important
for a proper SC lipid organization. It will be very interesting to locate the liquid
phase. In Section 6 we hypothesized that it is located in the central layer of the
long lamellar periodicity phase [93]. From the SC lipid models it became clear
that CER 1 (and also CER 4 in human skin) exerts a very specific influence on
both the lateral and lamellar lipid organization. The unsaturated long linoleate
moieties are most likely located in the intermediate layer, as a result of which at
least part of the lipid carbon chains will be in a fluid state in that region.
The ultimate goal is to formulate a molecular model for SC lipid organization,
since such a model is required to fully understand SC barrier function. Only then
may it become clear in detail which routes are followed by penetrants while
crossing the SC. Furthermore, such a molecular model will provide insight into
the mode of action of (trans) dermal drug delivery systems on the penetration
pathways and the reasons underlying the barrier impairment in the SC of
diseased skin, and human skin equivalents. Because the intercellular matrix is
such a heterogeneous mixture, enormous effort will be needed to accomplish this
work. Future studies on the permeability of SC lipid models [75, 94] and on SC
lipid organization, using WAXD, FTIR, ED, and maybe new techniques, like
AFM, are required to further unravel the complex molecular organization of SC

lipids that contribute to the unique barrier function of mammalian skin.
REFERENCES
1. Grayson S, Elias PM. Isolation and lipid biochemical characterization of stratum
corneum membrane complexes: Implications for the cutaneous permeability
barrier. J Invest Dermatol 1982; 78:128–135.
2. Yardley HJ, Summerly R. Lipid composition and metabolism in normal and
diseased epidermis. Pharmacol Ther 1981; 13:357–383.
3. Landmann L. The epidermal permeability barrier. Comparison between in vivo and
in vitro lipid structures. Eur J Cell Biol 1984; 33:258–264.
4. Wertz PW, Miethke MC, Long SA, Strauss JS, Downing DR. The composition of
the ceramides from human stratum corneum and from comedones. J Invest
Dermatol 1985; 84:410–412.
5. Landmann L. The epidermal permeability barrier. Anat Embryol 1988; 178: 1–13.
6. Hadgraft J. Recent developments in topical and transdermal delivery. Eur J Drug
Metab Pharmacokinet 1996; 21:165–173.
7. Madison KC, Swartzendruber DC, Wertz PW, Downing DT. Presence of intact
intercellular lipid lamellae in the upper layers of the stratum corneum. J Invest
Dermatol 1987; 88:714–718.
714.
9. Potts RO, Francoeur ML. The influence of stratum corneum morphology on water
permeability. J Invest Dermatol 1991; 96:495–499.
10. Fartasch M, Bassukas ID, Diepgen TL. Structural relationship between epidermal
lipid lamellae, lamellar bodies and desmosomes in human epidermis: an
ultrastructural study. Br J Dermatol 1993; 128:1–9.
11. Van der Meulen J, Van den Bergh BAI, Mulder AA, Mommaas AM, Bouwstra JA,
Koerten HK. The use of vibratome sections for the ruthenium tetroxide protocol: a
key for optimal visualization of epidermal lipid bilayers of the entire human stratum
corneum in transmission electron microscopy. J Microsc 1996; 184:67–70.
12. Elias PM, Menon GK. Structural and lipid biochemical correlates of the epidermal
permeability barrier. Adv Lipid Res 1991; 24:57–82.
13. Gray GM, White RJ. Glycosphingolipids and ceramides in human and pig
epidermis. J Invest Dermatol 1978; 70:336–341.
14. Wertz PW, Downing DT. Ceramides of pig epidermis: structure determination. J
Lipid Res 1983; 24:759–765.
15. Wertz PW, Downing DT. Epidermal lipids. In: Goldsmith LA, ed. Physiology,
Biochemistry, and Molecular Biology of the Skin. Oxford University Press, 1991;
24:205–235.
16. Robson KJ, Stewart ME, Michelsen S, Lazo ND, Downing DT. 6-Hydroxy-4-
sphingenine in human epidermal ceramides. J Lipid Res 1994; 35: 2060–2068.
17. Stewart ME, Downing DT. A new 6-hydroxy-4-sphingenine-containing ceramide
in human skin. J Lipid Res 1999; 40:1434–1439.
18. Long SA, Wertz PW, Strauss JS, Downing DT. Human stratum corneum polar
lipids and desquamation. Arch Dermatol Res 1985; 277:284–287.
19. Bouwstra JA, Gooris GS, Dubbelaar FER, Weerheim AM, IJzerman AP, Ponec M.
Role of ceramide 1 in the molecular organization of the stratum corneum lipids. J
Lipid Res 1998; 39:186–196.
20. Norlen L, Nicander I, Lundsjo A, Cronholm T, Forslind B. A new HPLC-based
method for the quantitative analysis of inner stratum corneum lipids with special
reference to the free fatty acid fraction. Arch Dermatol Res 1998; 290: 508–516.
21. Bloom M, Evans E, Mouritsen OG. Physical properties of the fluid lipidbilayer
component of cell membranes: a perspective. Q Rev Biophys 1991; 24: 293–397.
22. Corvera E, Mouritsen OG, Singer MA, Zuckermann MJ. The permeability and the
effect of acyl-chain length for phospholipid bilayers containing cholesterol: theory
and experiment. Biochim Biophys Acta 1992; 1107:261–270.
23. Lemmich J, Honger T, Mortensen K, Ipsen JH, Bauer R, Mouritsen OG. Solutes in
small amounts provide for lipid-bilayer softness: cholesterol, shortchain lipids, and
bola lipids. Eur Biophys J 1996; 25:61–65.
24. de Kruyff B, van Dijck PWM, Demel RA, Schuijff A, Brants F, van Deenen LLM.
Non-random distribution of cholesterol in phosphatidylcholine bilayers. Biochim
Biophys Acta 1974; 356:1–7.
25. Blaurock AE. Evidence of bilayer structure and of membrane interactions from x-
ray diffraction analysis. Biochim Biophys Acta 1982; 650:167–207.
26. Abrahamsson S, Dahlen B, Lofgren H, Pascher I. Lateral packing of hydrocarbon
chains. Prog Chem Fats Other Lipids 1978; 16:125–143.
27. Misell DL, Brown EB. Electron diffraction: an introduction for biologists. Practical
Methods in Electron Microscopy. Vol. 12. Amsterdam, Elsevier Science
Publishers, 1987.
28. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J
Biochem Physiol 1959; 37:911–917.
29. Ponec M, Weerheim A, Kempenaar J, Mommaas AM, Nugteren DH. Lipid
composition of cultured human keratinocytes in relation to their differentiation. J
Lipid Res 1988; 29:949–961.
30. Aly R, Shirley C, Cunico B, Maibach HI. Effect of prolonged occlusion on the
microbial flora, pH, carbon dioxide and transepidermal water loss on human skin. J
Invest Dermatol 1978; 71:378–381.
31. Bouwstra JA, Gooris GS, van der Spek JA, Bras W. Structural investigations of
human stratum corneum by small angle X-ray scattering. J Invest Dermatol 1991;
97:1005–1012.
32. White SH, Mirejovsky D, King GI. Structure of lamellar lipid domains and
corneocyte envelopes of murine stratum corneum. An X-ray diffraction study.
Biochemistry 1988; 27:3725–3732.
33. Bouwstra JA, Gooris GS, Vanderspek JA, Lavrijsen S, Bras W. The lipid and
protein structure of mouse stratum corneum: a wide and small angle diffraction
study. Biochim Biophys Acta 1994; 1212:183–192.
34. Bouwstra JA, Pilgram GSK, Gooris GS, Koerten HK, Ponec M. New aspects of the
skin barrier organization. Skin Pharmacol Appl Skin Physiol 2001; 14: 52–62.
35. Bouwstra JA, Gooris GS, Dubbelaar FER, Weerheim A, Ponec M. Cholesterol
sulfate and calcium affect stratum corneum lipid organization over a wide
temperature range. J Lipid Res 1999; 40:2303–2312.
36. Garson J, Doucet J, Lévêque J,Tsoucaris G. Oriented structure in human stratum

corneum revealed by X-ray diffraction. J Invest Dermatol 1991; 96:43–49.
37. Bouwstra JA, Gooris GS, Salomons-de Vries MA, Van der Spek JA, Bras W.
Structure of human stratum corneum as a function of temperature and hydration: a
wide angle X-ray diffraction study. Int J Pharm 1992; 84:205–216.
38. Gay CL, Guy RH, Golden GM, Mak VH, Francoeur ML. Characterization of low-
temperature (i.e. <65°C) lipid transitions in human stratum corneum. J Invest
Dermatol 1994; 103:233–239.
39. Naik A, Guy RH. Infrared spectroscopic and differential scanning calorimetric
investigations of the stratum corneum barrier function. In: Potts RO, Guy RH, eds.
Mechanisms of Transdermal Drug Delivery, New York, Marcel Dekker: 1997; 103:
87–162.
40. Pilgram GSK, Van Pelt AM, Spies F, Bouwstra JA, Koerten HK. Cryo-electron
diffraction as a tool to study local variations in the lipid organization of human
stratum corneum. J Microsc 1998; 189:71–78.
41. Pilgram GSK, Engelsma-Van Pelt AM, Bouwstra JA, Koerten HK. Electron
diffraction provides new information on human stratum corneum lipid organization
studied in relation to depth and temperature. J Invest Dermatol 1999; 113:403–409.
42. Bommannan D, Potts RO, Guy RH. Examination of stratum corneum barrier
function in vivoby infrared spectroscopy. J Invest Dermatol 1990; 95:403–408.
43. Bonte F, Saunois A, Pinguet P, Meybeck A. Existence of a lipid gradient in the
upper stratum corneum and its possible biological significance. Arch Dermatol Res
1997; 289:78–82.
44. Golden GM, McKie JE, Potts RO. Role of stratum corneum lipid fluidity in
transdermal drug flux. J Pharm Sci 1987; 76:25–28.
45. Bouwstra JA, Gooris GS, Bras W, Downing DT. Lipid organization in pig stratum
corneum. J Lipid Res 1995; 36:685–695.
46. Swartzendruber DC, Wertz PW, Kitko DJ, Madison KC, Downing DT. Molecular
models of the intercellular lipid lamellae in mammalian stratum corneum. J Invest
Dermatol 1989; 92:251–257.
47. Langner M, Hui SW. Dithionite penetration through phospholipid bilayers as a
measure of defects in lipid molecular packing. Chem Phys Lipids 1993; 65: 23–30.
48. Friedman MAJSJ. Lindane neurotoxic reaction in nonbullous congenital
ichthyosiform erythroderma. Arch Dermatol 1987; 123:1056–1058.
49. Lavrijsen APM, Oestmann E, Hermans J, Boddé HE, Vermeer BJ, Ponec M.
Barrier function parameters in various keratinization disorders: transepidermal
water loss and vascular response to hexyl nicotinate. Br J Dermatol 1993; 129:
50. Tupker RA, Pinnagoda J, Coenraads PJ, Nater JP Transepidermal Water Loss
Measurements by Means of an Evaporimeter, Springer-Verlag, Berlin, 1993; 123:
56–70.
51. Imokawa G, Kuno H, Kawai M. Stratum corneum lipids serve as a boundwater
modulator. J Invest Dermatol 1991; 96:845–851.
52. Yamamoto A, Serizawa S, Ito M, Sato Y. Stratum corneum lipid abnormalities in
atopic dermatitis. Arch Dermatol Res 1991; 283:219–223.
53. Di Nardo A, Wertz P, Giannetti A, Seidenari S. Ceramide and cholesterol
composition of the skin of patients with atopic dermatitis. Acta Dermatol Venereol
(Stockh) 1998; 78:27–30.
54. Lavrijsen APM, Bouwstra JA, Gooris GS, Weerheim A, Boddé HE, Ponec M.
Reduced skin barrier function parallels abnormal stratum corneum lipid
organization in patients with lamellar ichthyosis. J Invest Dermatol 1995; 105: 619–
624.
55. Rajka G. Transepidermal water loss on the hands in atopic dermatitis. Arch
Dermatol Forsch 1974; 251:111–115.
56. Wirth H, Gloor M, Stoika D. Sebaceous glands in uninvolved skin of patients
suffering from atopic dermatitis. Arch Dermatol Res 1981; 270: 167–169.
57. Bleck O, Abeck D, Ring J. Two ceramide subfractions detectable in CER(As)
position by HPTLC in skin surface lipids of non-lesional skin of atopic eczema. J
58. Bouwstra JA, Gooris GS, Cheng K, Weerheim A, Bras W, Ponec M. Phase
behavior of isolated skin lipids. J Lipid Res 1996; 37:999–1011.
59. Bouwstra JA, Gooris GS, Dubbelaar FER, Weerheim AM, Ponec M. pH,
cholesterol sulfate and fatty acids affect the stratum corneum lipid organization. J
Invest Dermatol Symp Proc 1998; 3:69–74.
60. Pilgram GSK, Engelsma-van Pelt AM, Oostergetel GT, Koerten HK, Bouwstra JA.
Study on the lipid organization of stratum corneum lipid models by (cryo-) electron
diffraction. J Lipid Res 1998; 39:1669–1676.
61. Bouwstra JA, Dubbelaar FER, Gooris GS, Weerheim AM, Ponec M. The role of
ceramide composition in the lipid organization of the skin barrier. Biochim
Biophys Acta 1999; 1419:127–136.
62. Xiang TX, Anderson BD. Permeability of acetic acid across gel and
liquidcrystalline lipid bilayers conforms to free-surface-area theory. Biophys J
1997; 72:223–237.
63. OgisoT, Hirota T, Iwaki M, Hino T, Tanino T. Effect of temperature on
percutaneous absorption of terodiline, and relationship between penetration and
fluidity of the stratum corneum lipids. Int J Pharm 1998; 176:63–72.
64. Pilgram GSK, Vissers DCJ, van der Meulen H, Pavel S, Lavrijsen APM, Bouwstra
JA, Koerten HK. Aberrant lipid organization in stratum corneum of patients with
atopic dermatitis and lamellar ichthyosis. J Invest Dermatol 2001; 117:710–717.
65. Fartasch M, Bassukas ID, Diepgen TL. Disturbed extruding mechanism of lamellar
bodies in dry noneczematous skin of atopics. Br J Dermatol 1992; 127: 221–227.
66. Ghadially R, Williams ML, Hou SYE, Elias PM. Membrane structural
abnormalities in the stratum-corneum of the autosomal recessive ichthyoses. J
67. Fartasch M. Epidermal barrier in disorders of the skin. Microsc Res Tech 1997; 38:
361–372.
68. Niemi KM, Kanerva L, Kuokkanen K. Recessive ichthyosis congenita type II. Arch
Dermatol Res 1991; 283:211–218.
69. Hohl D, Huber M, Frenk E. Analysis of the cornified cell envelope in lamellar
ichthyosis. Arch Dermatol 1993; 129:618–624.
70. Paige DG, Morsefisher N, Harper JI. Quantification of stratum corneum ceramides
and lipid envelope ceramides in the hereditary ichthyoses. Br J Dermatol 1994; 131:
23–27.
71. Huber M, Rettler I, Bernasconi K, Frenk E, Lavrijsen APM, Ponec M, Bon A,
Lautenschlager S, Schorderet DF, Hohl D. Mutations of keratinocyte
transglutaminase in lamellar ichthyosis. Science 1995; 267:525–528.
72. Sato J, Denda M, Nakanishi J, Nomura J, Koyama J. Cholesterol sulfate inhibits

proteases that are involved in desquamation of stratum corneum. J Invest Dermatol
1998; 111:189–193.
73. Cheetham JJ, Chen RJ, Epand RM. Interaction of calcium and cholesterol sulphate
induces membrane destabilization and fusion: implications for the acrosome
reaction. Biochim Biophys Acta 1990; 1024:367–372.
74. Kitson N, Monck M, Wong K, Thewalt J, Cullis P. The influence of cholesterol 3-
sulphate on phase behaviour and hydrocarbon order in model membrane systems.
Biochim Biophys Acta 1992; 1111:127–133.
75. Kuempel D, Swartzendruber DC, Squier CA, Wertz PW. In vitro reconstitution of
stratum corneum lipid lamellae. Biochim Biophys Acta 1998; 1372: 135–140.
76. Bouwstra JA, Gooris GS, Dubbelaar FER, Ponec M. Phase behavior of lipid
mixtures based on human ceramides: coexistence of crystalline and liquid phases. J
Lipid Res 2001; 42:1759–1770.
77. Bouwstra J, Gooris GS, Dubbelaar FER, Ponec M. Phase behavior of stratum
corneum lipid mixtures based on human ceramides: the role of natural and
synthetic ceramide 1. J Invest Dermatol (2002); 118:606–617.
78. Carruthers A, Melchior DL. A study of the relationship between bilayer water
permeability and bilayer physical state. Biochemistry 1983; 22:5797–5807.
79. Dahlén B, Pascher I. Molecular arrangements in sphingolipids. Thermotropic phase
behaviour of tetracosanoylphytosphingosine. Chem Phys Lipids 1979; 24:119–133.
80. Norlén L. Skin barrier structure and function: the single gel phase model. J Invest
Dermatol 2001; 117:830–836.
81. Singer SJ, Nicolson GL. The fluid mosaic model of the structure of cell
membranes. Science 1972; 175:720–731.
82. Brown RE. Sphingolipid organization in biomembranes: what physical studies of
model membranes reveal. J Cell Sci 1998; 111:1–9.
83. Verkleij AJ. Lipid intramembrane particles. Biochim Biophys Acta 1984; 779: 43–
63.
84. Tocanne JF, Cezanne L, Lopez A, Piknova B, Schram V, Tournier JF, Welby M.
Lipid domains and lipid/protein interactions in biological membranes. Chem Phys
Lipids 1994; 73:139–158.
85. Welti R, Glaser M. Lipid domains in model and biological membranes. Chem Phys
Lipids 1994; 73:121–137.
86. Liu F, Sugar IP, Chong PLG. Cholesterol and ergosterol superlattices in three-
component liquid crystalline lipid bilayers as revealed by dehydroergosterol
fluorescence. Biophys J 1997; 72:2243–2254.
87. Virtanen JA, Cheng KH, Somerharju P. Phospholipid composition of the
mammalian red cell membrane can be rationalized by a superlattice model. Proc
Natl Acad Sci USA 1998; 95:4964–4969.
88. Krill SL, Knutson K, Higuchi WI. The stratum corneum lipid thermotropic phase
behavior. Biochim Biophys Acta 1992; 1112:281–286.
89. Pilgram GSK, van der Meulen J, Gooris GS, Koerten HK, Bouwstra JA. The
influence of two Azones and sebaceous lipids on the lateral organization of lipids
isolated from human stratum corneum. Biochim Biophys Acta 2001; 1511:244–
254.
90. Nemanic MK, Elias PM. A novel cytochemical technique for visualization of
permeability pathways in mammalian stratum corneum. J Histochem Cytochem
1980; 28:573–578.
91. Bowser PA, White RJ. Isolation, barrier properties and lipid analysis of stratum
compactum, a discrete region of the stratum corneum. Br J Dermatol 1985; 112:1–
14.
92. Boddé HE, van den Brink I, Koerten HK, de Haan FHN. Visualization of in vitro
percutaneous penetration of mercuric chloride; transport through intercellular space
versus cellular uptake through desmosomes. J Controlled Release 1991; 15:227–
236.
skin barrier. Acta Dermatol Venereol Suppl (Stockh) 2000; 208: 23–30.
94. Norlén L, Engblom J. Structure-related aspects on water diffusivity in fatty acid-
soap and skin lipid model systems. J Controlled Release 1998; 63: 213–226.
95. Pilgram GSK, Engelsma-van Pelt AM, Ponec M, Bouwstra JA, Koerten HK. The
application of (cryo-)electron diffraction to study the lateral organization of human
stratum corneum lipids: an overview. Res Adv Lipids 2000; 1: 119–131.
7
The Mammalian Skin Barrier: Structure,
Function, and Formation Considerations
Lars Norlén
University of Geneva, Geneva, Switzerland
Since most, if not all, natural processes are irreversible, structural order in
biological systems intimately depends on how this order is created [[1], pp. 17–
72]. Thus considerations of formation and of structure-function can hardly be
separated. This is the framework within which the “membrane-folding” [2] and
“single gel phase” model [3, 4] for skin barrier structure, function, and formation
were conceived. Also, the tremendous increase in our knowledge of the
complexity of the skin barrier over the last 25 years has, in my opinion, further
served to underline the need for conceptual tools to make this information more
accessible to critical interpretations. Some arguments to justify this statement are
enumerated at the outset.
1. From a thermodynamic point of view, biological systems are ex hypothesi
out of equilibrium; that is, all natural processes are irreversible. Further,
biological systems are open to the flux of energy and matter; they are usually far
from equilibrium, nonlinear in their flux-force relationships, characterized by and
strong coupling between their processes. Also, since most, if not all, biological
systems exhibit gradients (in e.g., energy density) over distances of less than 10
Å, they cannot have “local thermodynamics.” Consequently, they are not at
“local equilibrium” but usually are stabilized by secondary or higher minimum
energy order steady states [[1], pp. 17–72].
These considerations (i.e., that biological organizations can maintain
themselves only if there is a flow of energy and that this flow requires the system
to be out of equilibrium) complicate severely the biological interpretation of
experimental in vitro and in vivo data. Structural order comes from the existence
of constraints. In any in vitro (and even in vivo) experimental setup some or all of
these constraints may differ from those of the endogenous, living situation. If
this is not acknowledged, structural-functional data obtained under in vitro
conditions may, of course, be grossly misleading.
2. The need for conceptual models is also apparent from the repeated reports
of the dominance of orthorhombic crystalline order [5, 6] in a system (in this
case the skin barrier membrane structure) that contains more than 30% (wt/wt)
cholesterol (~40–45 wt % if the cholesteryl esters are included) [7, 8]. If
cholesterol is inserted between lipid hydrocarbon chains, no true orthorhombic
or hexagonal crystals can be formed; that is, under these conditions, no
140 NORLÉN
orthorhombic or hexagonal wide-angle reflections can be obtained from X-ray

experiments. Consequently, either cholesterol is inhomogeneously distributed in
the stratum corneum intercellular lipid matrix or our crystallographic data do not
reflect the structural organization of the true non equilibrium situation in vivo.
Additional crystallographic examples are the reported presence of anhydrous
cholesterol crystals in the intercellular space of stratum corneum [5, 6], reports
of lipid crystallites larger than about 0.3 µm [5], the reported large experimental
as well as interindividual variability in stratum corneum diffraction patterns [5, 6],
and the reported large difference between the in vivo and the ex-vivo preparations
in the relative amount of hexagonal/orthorhombic chain packing at 32 °C (i.e.,
normal skin temperature) [9].
3. The outcome of experiments using mixtures of synthetic or extracted skin
lipids as models of skin barrier structural organization ultimately depends on the
chosen lipid composition. Other factors may, however, also be of critical
importance. These include the amount of water associated with the lipid head
groups, the pH locally over the membrane surface, the amount of mono- and
multivalent ions, and the amount of gases (e.g., CO2, O2) present. To date, from a
biophysical perspective, none of these factors have been satisfactorily
characterized in the endogenous stratum corneum. Another serious shortcoming
of lipid model mixture experiments is that in vivo gradients, in, say, the
chemical potential of water, pH, ion concentrations, lipid composition, gas
concentrations (e.g., CO2, O2), and heat flows over the stratum corneum lipid
membranes, as well as chemical reactions associated with the membrane surface,
are difficult to model in vitro. Consequently a straightforward interpretation of
the outcome of skin lipid model mixture experiments with respect to the situation
in vivo may be most difficult to reach.
4. The literature of skin barrier lipid analysis is full of contradictory data [10].
This is largely due to different preparation techniques used and to the use of
moderately rigorous experimental designs and statistical evaluations. In addition,
skin lipid analysis is particularly prone to contamination [11, 8, 10, 11].
5. Morphological skin data are often contradictory and confusing. One
example is “lamellar bodies,” of which a plethora of forms, numbers, sizes and
appearances have been reported (all are not lamellar, but many appear empty or
partly lamellar, contain “onionlike” structures, have a granular appearance, or
exhibit “abnormal internal contents”) [12, 13]. These discrepancies are certainly
partly due to artifacts introduced by chemical fixation and dehydration during
sample preparation for classical electron microscopy.
6. Qualified economizing in energy is a characteristic feature of most
biological systems. Thus, during biological processes energy dissipation is likely
to be kept at an absolute minimum through optimal design of transition matrices.
One may therefore presume that the entities that are taking part in a biological
process (e.g., a structural membrane transformation or an enzymatic action) act
in a highly cooperative manner. This implies that stochastic processes, which
rely on random encounters, in most cases are not involved, since in vivo
THE MAMMALIAN SKIN BARRIER 141
processes must depend on limited supplies of energy and must be accomplished

during extremely narrow time spans. For obvious reasons these restrictions are
often difficult to take into account during in vitro modeling.
1
DIFFICULTIES CONFRONTING SKIN BARRIER
RESEARCH
To make predictions about the structure, function and dynamics of a biological
system (e.g., the mammalian skin barrier) the system composition and topology,
as well as any gradients over the system, ought to be known in considerable
detail. Skin barrier research is here confronted with serious difficulties. This is
not only because precise, uncontaminated compositional data are difficult to
obtain, as noted in introductory item 5, but also because human skin separates two
very different compartments (i.e., inside and outside of the organism) and
consequently several pronounced gradients are present over the skin. To
complicate matters even further, the composition as well as the gradients are
likely to be inhomogeneously distributed because the tissue features a complex
three-dimensional compartmentalization.
The most evident gradient over the skin barrier membrane system is in the
chemical potential of water, with a water concentration of approximately 70 to
80% (w/w) of the living tissue inside the body and a variable water concentration
of about 10 to 20% (w/w) of the outer stratum corneum in equilibrium with the
environmental air, which is often dry. Less is known about remaining gradients
and distributions over the skin of importance for lipid phase behavior, such as
ion concentrations, pH, gas concentrations (e.g., CO2, O2), lipid compositions,
and temperature. One contributing reason may be the scarcity of topological skin
data. In fact, the only incontestably known structural features of the stratum
corneum skin barrier lipid matrix are that it is extracellular and multilamellar
[14, 15] and, at physiological conditions, it is largely “crystalline” [5, 6, 16].
Little is known about the actual lamellar organization, the local distribution of
water, ions, and gases, the association of proteins, or the connectivity or genus
(number of holes) of the continuous (?) membrane structure.
Nevertheless, the data available may suffice to allow some far-reaching
speculations to be made not only with respect to skin barrier structure and
function, but also with respect to the formation of this highly specialized
membrane structure.
The recently published membrane-folding model [2] postulates that skin
barrier formation may take place as a continuous and highly dynamic unfolding
of a single and coherent hyperbolic lipid structure with symmetry into a flat
parabolic lipid structure with a concomitant “crystallization” (i.e., close packing
or “condensation”) of the emerging multilamellar lipid structure representing the
developing skin barrier. It was further proposed in the single gel phase model [3, 4]
that the skin barrier (i.e., the intercellular lipid within the stratum corneum)
142 NORLÉN
exists as a single and coherent “gel” structure, or equivalently an extremely

tightly packed liquid crystal with much restricted lateral lipid diffusivity, which,
however, may express true crystalline hydrocarbon chain packing in local
cholesterol-deficient regions (Fig.1).
2
SKIN BARRIER FORMATION
In 1986 Landmann presented a model for the formation of the mammalian skin
barrier, the multilamellar lipid matrix of the stratum corneum intercellular space
[17]. The basic features of the Landmann model are as follows:
1. Formation of vesicles termed “lamellar bodies”* via membrane fission, or

budding, from the trans-Golgi network of epidermal keratinocytes [18]. The
“lamellar bodies” are visualized as discrete unilamellar vesicles containing
in their turn multiple stacked flattened unilamellar vesicles, termed “lamellar
body-disks.”
2. Targeted diffusion of “lamellar bodies” in the cytosol of the stratum
granulosum cells from the trans-Golgi network to the stratum granulosum—
stratum corneum interface.
3. Fusion of “lamellar bodies” with each other and with the plasma membrane
of the stratum granulosum cells at the stratum granulosum—stratum
corneum interface [18].
4. Fusion of “lamellar body-disks” into continuous multilamellar membrane
sheets in the intercellular space at the stratum granulosum—stratum corneum
interface.
However, there remain some unresolved questions about the Landmann model
that may be summarized in the following way: Why would nature disrupt a
continuous membrane structure (the trans-Golgi network) to form another
continuous membrane structure (the intercellular lipid matrix of the stratum
corneum), especially since this would cost energy (initially budding and then two
consecutive fusion processes), imply decreased control (due to membrane
disintegration) over the barrier formation process (including water
compartmentalisation and control hereof), and be time-consuming (budding,
diffusion, and then fusion)?
Sections 2.1 to 2.7 present an alternative model for skin barrier morphogenesis
based on the concept of intersection-free membrane folding (i.e., ignoring
fusion) [24].
*Synonyms: lamellar granules, membrane-coating granules, Odland bodies [12, 13, 17,
18–23].
FIGURE 1 Schematic view of the skin: (a) full skin thickness; (b) epidermis; (c) horny layer (stratum corneum, s.c.) and uppermost part of
granular layer (stratum granulosum, s.g.); (d) stacked lipid lamellae of the intercellular space of stratum corneum, constituting the actual skin
barrier; (e) main crystalline lipid hydrocarbon chain packing (hexagonal) of cholesterol-deficient areas of the skin barrier; (f) interzone between
stratum granulosum and stratum corneum where skin barrier formation takes place; (g) cubic membrane structure proposed to be responsible for
normal skin barrier formation; (h) liquid crystalline character of the curved lipid bilayer constituting the cubic membrane.
THE MAMMALIAN SKIN BARRIER
143
144 NORLÉN
2.1
The Membrane-Folding Model
Homeostasis, the maintenance of a semi static physiological condition (usually
not an equilibrium condition but a feature of a secondary, or higher, minimum
energy order), is a highly dynamic series of events including membrane
morphogenesis [[25, 26]; [27], pp. 167–171]. Since membrane continuity is
mandatory for barrier capacity in biological systems (e.g., cell integrity, skin
barrier function), surface continuity should be preserved, and occurrence of
fusion processes minimized, during membrane formation [[24]; [28], pp. 317–
321]. One way of achieving this is by intersection-free membrane folding [25]. It
is therefore natural to explore the possibility that skin barrier morphogenesis may
depend on such a mechanism.
Fusiogenic processes (e.g., membrane fusion, exocytosis, endocytosis) require
extensive morphological changes, at least locally and transiently, to highly
curved surfaces (which imply high bending energy, as suggested in Refs. [29]
and [30]) [31]. Further, fusiogenic processes can be questioned on general
ground because the lipids claimed to be most fusiogenic (e.g., lysolecithin,
monoolein) do not promote flat bilayer organization, but, quite the contrary, are
characterized by an intrinsic high curvature [[27], p. 17; 32]. Suggestions that the
self-assembled geometries favored by these fusiogenic lipids (i.e., micelles and
inverted micellar morphologies, respectively) mirror intermediate structures of
membrane fusion have been disposed of [33]. Instead, stalks, which combine the
qualities of minimum size and compound curvature, have been identified as the
lowest energy membrane intermediates in fusion [34]. The calculated stalk
energies are still comparatively high, however, and thus a dilemma remains [35].
The Landmann model for skin barrier formation [17] involves four fusiogenic
processes (one budding and three fusion processes). Another troubling feature
with this model is the local high curvature of the “lamellar body-disk” edges.
This not only supposes the presence of lipids with liquid like hydrocarbon chains
(i.e., short and/or unsaturated) but also curvature-induced segregation of these
liquid crystal forming lipids [36, 37] and, consequently, two-dimensional phase
separation, within the “lamellar body-disks.” However, other possibilities remain,
such as lipid-protein interactions at the “lamellar body-disk” edges.
In fact, through intersection-free membrane folding and unfolding, the whole
process of skin barrier formation could, theoretically, be performed without
energetically unfavorable processes like membrane fission (i.e., budding) and
membrane fusion, without two-dimensional phase separation, and without
vesicular diffusion (i.e., slow transport, lasting for hours) of lipid components.
It has therefore been proposed that skin barrier morphogenesis may take place
via a continuous, highly dynamic process of “intersection-free unfolding” (i.e., a
continuous deformation of a single and coherent three-dimensional* lipid
structure into a flat two-dimensional† lipid structure) with a concomitant
“crystallization” of the developing stacked multilamellar lipid structure
representing the skin barrier [2], In fact, continuous, dynamic foldings and
unfoldings between cubic (three-dimensionally hyperbolic) and lamellar (two-
dimensionally Euclidian) structures are present in other biological systems, such
as the endoplasmatic reticulum of metazoan cells [[27], p. 94; [38, 39]], and the
thylakoid membrane of plant chloroplasts [[27], pp. 140–141; [40–42].
Further, coherence between three-dimensional (e.g., cubiclike) and two-
dimensional (i.e., lamellar) membrane morphologies does not imply local high
bending energy because the proposed folded continuous three-dimensional
structure with symmetry is likely to express oscillating periodicity [[43–46] pp.
177–190]. This is not possible for a flat two-dimensional entity like a “lamellar
body-disk.” Thus, during unfolding, the local “edge-curvature” between folded
cubiclike and lamellar regions of the symmetrical continuous three-dimensional
structure proposed here could be visualized as part of a standing wave motion
[45]. Accordingly, phase separation of lipids is not implied (cf. outer lamellar
membranes of cubosomes) [44, 45].
The proposed symmetrical single and coherent three-dimensional lipid
structure expressing this intersection-free folding and unfolding may have either
a cubic lattice [47, 48] or a rhombohedral arrangement, since, in vitro, such
intermediate phases are stabilized by increased hydrocarbon chain length and
degree of saturation* [cf. [31, 49, 50]]. It should be noted that cubic lipid phases
may represent, in the scheme of infinite periodic minimal surfaces (IPMS), three-
periodic minimal surfaces [[51], pp. 50–55] and may therefore be transformed to
flat lamellar membrane morphologies with comparatively small bending energy
cost (i.e., the transition involves only a small deviation from the preferred
molecular surfactant parameter, v/al, of the membrane lipids) (V=volume,
a=area, l=effective hydrocarbon chain length). This close geometrical
resemblance between lamellar (L ) and bicontinuous cubic (V1, V2) lipid
morphologies is emphasized by the low enthalpy difference between these two
phases (approximately 0.5 kJ/mol lipid) [52], compared with the enthalpy
difference between the lamellar and reversed hexagonal (HII) phases (~5–10 kJ/
mol lipid) [53]. Further, cubic membranes representing cubic three-periodic
minimal surfaces of genus three (G, D, and P surfaces) can be formed with the
*That is, a membrane constellation centered around a surface with negative average
Gaussian curvature, or equivalently with genus higher than 1 (i.e., containing 2 or more
handles or holes).
†That is, a membrane constellation centered around a surface with zero average Gaussian
curvature, or equivalently with genus 1 (i.e., containing 1 handle or hole).
146 NORLÉN
least energy cost because they are the most homogeneous (i.e., they have the
smallest Gaussian curvature inhomogeneity over the whole structure) [[28], p.
151]. However, rhombohedral distortions of the D and P surfaces can occur at only
slightly higher bending energy costs [[28], p. 159]. Also, geometric analysis
indicates that hyperbolic lipid membrane structures (e.g., cubic) are most likely
to be found in lipid systems that form lamellar phases readily [[28], p. 169].
The proposed single and coherent three-dimensional membrane structure may
be multilayered; that is, it may express parallel multimembranous foldings* with
cubiclike symmetry, have a large lattice parameter of, say, 50 to 500 nm [[19, 28],
pp. 317–323] and develop from the trans-Golgi network of the epidermal cells
through intersection-free folding [2].
Since the composition of cell space is nonstatic, the multilamellar lipid
structure that is formed may be stabilized by steady state conditions (i.e., gradients)
rather than by thermodynamic equilibrium conditions. In addition, the absence of
geometric constraints would allow the unfolding process to be extremely fast
(i.e., instantaneous), since it basically represents a phase† transformation from
cubiclike to lamellar morphology. In as much as the thermodynamics of this
unfolding procedure is related to curvature energy, asymmetrical objects (e.g.,
proteins) that bind to both sides of the lipid bilayer could play a regulatory role
[54]. The procedure may thus be finely tuned by subtle stimuli. Skin barrier
formation taking place via an unfolding process may consequently be highly
controlled and very fast (in comparison to, e.g., diffusion-dependent processes,
Ref. [17]) [2].
2.2
Visualisation of Cubiclike Membrane Morphologies
The difficulty involved in visualizing the barrier-forming process proposed here
is obvious. Partly because of the sensitivity to sample preparation (e.g.,
dehydration, organic solvent exposure, fixation) of the proposed folded three-
dimensional lipid structure with symmetry and partly because micrographs
obtained are likely to become blurred if the lattice parameter of the unit cell of this
three-dimensional lipid structure is smaller than the section thickness. Therefore,
the seemingly discrete “lamellar bodies” observed in electron micrographs may
represent local lamellar crystallizations of a single continuous three-dimensional
lipid structure, where the three-dimensional (i.e., cubiclike) parts are largely
blurred [2]. This is because owing to their lamellar two-dimensional symmetry
and probably more crystalline morphology, the lamellar parts of the proposed
three-dimensional structure should be less sensitive to sample preparation and
*Stratum corneum lipid composition is characterized by the domination of lipids with

saturated, very long hydrocarbon chains [7, 8, 11], which to some degree may mirror the
lipid hydrocarbon chain distributions of stratum granulosum.
more easily visualized in electron micrographs, In fact, the repeat distance of the
lamellae of the “lamellar bodies” (64–70 Å), corresponds closely to the repeat
distance of the crystalline intercellular lamellar lipid matrix of the stratum
corneum (64 Å) [16]. Further, blurring of the three-dimensional parts of the
proposed continuous membrane structure could partly explain the reported
plethora of forms, numbers, sizes, and appearances of “lamellar bodies” [13, 13].
2.3
Crystallization
The proposed “crystallization” of the emerging extracellular lamellar lipid
structure, taking place at the border between stratum granulosum and stratum
corneum, represents a change from a less ordered (i.e., liquid crystalline) into a
more ordered (i.e., “gel”) state.* The driving force for this “crystallization” (i.e.,
phase change from liquid crystal to “gel”) could be the drastically lowered water
content [[55]; [56], pp. 440–443] and/or the dramatic change in lipid composition
[[25, 26]; [28], pp. 215–218; [57]] known to occur at this border zone. In fact, at
the border between stratum granulosum and stratum corneum the total tissue
water content decreases momentarily from about 70 to 40% (w/w) [58, 59]. At
the same site, whereas initially the lipid composition was dominated by medium-
chain mono- and diunsaturated phospholipids (mainly C16:0, C18:l, and C18:2)
and glycosylceramides (mainly C16:0, C18:l, C18:2 and C24:0-C28:0) [61, 61]
in the stratum granulosum these lipid species are virtually absent in the stratum
corneum [62–64]. In addition, the cells of upper stratum granulosum are enriched
in certain acid hydrolases including phospholipase A and glycosidase [65–67].
One might speculate that the decreased head group polarity of the ceramides, due
to cleavage of the sugar moieties of their precursors the glycosylceramides taking
place at the stratum granulosum-stratum corneum interface, initialize and
provides the driving force for the “crystallization” process†. Further, if only
limited lateral diffusion of lipids takes place during this “crystallization,” the
transition from liquid crystal to “gel” may be almost momentary (i.e.,
milliseconds) [2].
Phase coexistence between liquid crystalline and “gel” domains may not be
expected to occur even during the actual transition. This is because, for relatively
uncharged monocompositional lipid membranes, thermodynamic fluctuations‡
near the L -gel phase transition may not be intense enough to approximate the
formation of a domain of the opposite phase. This rules out the existence of even
loosely defined solid domains in a fluid phase, leading to complete smearing of
*In fact, multilamellar foldings with cubiclike symmetry have been observed in many
different biological systems [27].
†Note that the current use of the word “phase” in a biological context does not necessarily
reflect a thermodynamically stable phase state.
148 NORLÉN
the transition [68, 69]. Further, the large relative amount of cholesterol in the
stratum corneum lipid matrix (~30 mol %) may counteract an abrupt increase in
permeability near the liquid crystalline to “gel” phase transition [70–72].
2.4
Flip-Flop from Hairpin to Splayed-Chain Conformation?
If a part of the ceramides of the skin barrier exists as a monolayer in the splayed-
chain conformation in the mature “crystallized” skin barrier, a flip-flop from
hairpin to splayed-chain conformation must take place in this fraction during skin
barrier formation. Generally flip-flop is very slow for membrane lipids (e.g.,
phosphatidylcholine, sphingomyelin, glycosylceramides), since it is energetically
highly disadvantageous to break the interactions between the hydrophilic head
groups of neighboring molecules and water, respectively, or alternatively to drag
head group-associated water through the hydrophobic hydrocarbon chain matrix.
However, for ceramides, which in the liquid crystalline state typically bind very
little water (0–1 water molecule per lipid molecule) [73], in comparison to their
precursors the glycosylceramides, which bind about 5 to 10 water molecules per
cerebroside [74, 75], a flip-flop movement ought to be considerably faster.
During dehydratization [59] and deglycosylation [66] at the stratum granulosum-
stratum corneum interface, it is not unlikely that a part of the now much less
hydrated ceramides could undergo reorganization from the hairpin into the
splayed-chain conformation. This is because the ceramides still contain some
rather potent polar groups, namely two to four O—H groups and one amide N—
H group as proton donators and carbonyl (C=O:) and hydroxyl (H—O:) oxygens
as proton acceptors, which could form a lateral H-bonding system in the center
of a monolayer and thus stabilize the splayed-chain organization of the
“crystalline” membrane. This is in fact the case for crystals of phytosphingosine-
based ceramides, where the monolayer organization is energetically more
favorable than the corresponding bilayer organization [76]. The monolayer
organization may be preferred over the bilayer organization for
phytosphingosine-based ceramide crystals because the stabilizing H-bonding
system between the two hydrocarbon matrices is better preserved when it is
shielded from the competition of water. Also, the dominance of saturated long
fatty acid chains may further stabilize the monolayer conformation because there
*Note that because as much as 30 mol% of cholesterol may be present, this more ordered
state, the “gel” state, does not necessarily express typical hexagonal wide-angle
reflections (4.15 Å). Instead, it may be regarded as an extremely tightly packed liquid
crystal, with its lipid lateral diffusivity much restricted or even nonexistent.
†Inhibition of -glycocerebrosidase has been reported to induce a “perturbed” skin barrier
membrane structure and a decreased barrier function [66, 67].
‡Thermodynamic fluctuations are responsible for the experimentially measured specific
heat and lateral compressibility anomalies near the liquid crystalline-gel transition [77, 78].
is a better match of hydrocarbon chains in a splayed-chain conformation. The

presence or absence in the endogenous stratum corneum of ceramides in the
splayed-chain conformation thus remains an open question.
2.5
Phase Separation?
Crystallization of the intercellular lipid matrix at the border zone between stratum
granulosum and stratum corneum may not only induce a splayed-chain
conformation of part of the ceramide fraction (i.e., the phytosphingosine- and
dihydrosphingosine-based ceramide fractions (Table 1), since ceramides with
trans-4-5-unsaturated sphingosine more easily pack in the hairpin conformation
as suggested in a personal communication by Prof. Irmin Pascher, but also
induce a lateral and/or vertical segregation of lipid components that do not fit in
the tightly packed monolayer hydrocarbon matrix. Consequently, different lipid
species could be enriched in different matrices of the layered crystalline lipid
structure. One important example is cholesterol, which may not be evenly
distributed in the lateral and/or vertical dimension. However, an uneven
distribution of cholesterol does not automatically mean that a true phase
separation is present, since the system may still from a macroscopic
thermodynamic point of view behave as one single and coherent phase. An
example of this phenomenon is the ripple dimyristoyl phosphatidylcholine-
cholesterol bilayer, which can be described as a single phase even though
cholesterol is not evenly distributed in the structure [79–80]. Further, high
cholesterol content and saturated long hydrocarbon chains (typical of skin barrier
lipid composition) favor the dissipation of domain line boundaries, bringing
phosphatidylcholine monolayers (in hairpin configuration at the air-water
interface) from two-phase coexistence to what is apparently a one-phase system
[81]. In short, nonrandom microscopic distribution of molecules (e.g.,
cholesterol) may occur even if the system macroscopically (thermodynamically)
behaves as a single phase. However, the permeability properties of a “single
phase” with non random microscopic distribution of lipid molecules may still
largely depend on the local lipid organization/segregation at the molecular level
cf. Ref. [82].
TABLE 1 Mean Number of Carbons and Double Bonds per Alkyl Chain of Stratum Corneum Lipids from Epidermal Cystsa
150 NORLÉN
152 NORLÉN
2.6
Recent Experimental Support
In fact, support for the notion that skin barrier formation may take place via a
continuous unfolding of intersection-free membrane is the recent finding of an
“extensive intracellular tubulo-reticular cisternal membrane system within the
apical cytosol of the outermost stratum granulosum” [18]. This membrane system
is reported to be composed of a widely disbursed transGolgi-like network
associated with arrays of contiguous lamellar bodies and deep invaginations, or
honeycomb extensions, of the stratum granulosum-stratum corneum interface
[18]. Direct membrane connections between “lamellar bodies” themselves,
between the trans-Golgi network and lamellar bodies, and between “deep
invaginations” in the stratum granu losum cells and the “envelope” of adjacent
“lamellar bodies” are identified [18]. In 1998 Elias et al. [18] suggested that
“lamellar bodies” are formed by budding (i.e., membrane fission) from the
cisternae of the trans-Golgi-like network and that the “deep invaginations” of the
stratum granulosum-stratum corneum interface are formed as a result of lamellar
body fusion. However, an alternative interpretation is that neither membrane
fission nor membrane fusion takes place during skin barrier formation, but that
intersection-free membrane folding and unfolding is responsible for skin barrier
morphogenesis. This implies that the trans-Golgi network and lamellar bodies of
the uppermost stratum granulosum cells, as well as the multilamellar lipid matrix
of the intercellular space at the border zone between stratum granulosum and
stratum corneum could represent one and the same continuous membrane
structure [2].
2.7
Tentatively Proposed Detailed Description of the Barrier-
Forming Process
The following detailed description of the proposed barrier-forming lipid structure
is but one of many possible, all of which share the basic idea of three-
dimensional symmetry, continuity, and dynamics. Tentatively, the proposed
single and coherent three-dimensional lipid structure may consist of two
components: (1) an outer folded membrane (e.g., cubiclike) with a large (>500
nm) lattice parameter corresponding both to the trans-Golgi-like network and the
honeycomb extensions (i.e., the extensive intracellular tubulo-reticular cisternal
membrane system [18] as well as to the outer “limiting membrane” of the
lamellar bodies of the outermost stratum granulosum cells, and (2) an inner more
highly curved membrane with symmetry expressing continuous foldings and
unfoldings between cubiclike and lamellar structures [2]. This inner more highly
folded membrane should correspond to the lamellar, or nonlamellar, interior (i.e.,
the lipid content) of lamellar bodies and deep invaginations, as well as to the
intercellular stacked lamellar, or nonlamellar, lipid matrix at the intercellular

space between stratum granulosum and stratum corneum. The outer folded
membrane (or parallel multimembranous foldings) of this single, continuous
lipid structure may thus function as a space divider, to separate the intercellular
space from the interior of the stratum granulosum cell, while the inner folded
membrane (or parallel multimembranous foldings) of the same lipid structure
may more directly, through lamellar unfolding, be responsible for the formation
of the multilamellar lipid matrix of the stratum corneum intercellular space [2].
The continuous change from inner periodicity to outer shape of the proposed
single and coherent three-dimensional membrane structure with sym metry could
have components of standing wave oscillations [[45]; [46], pp. 177–190]. This
implies that the newly formed unfolded stacked lamellar lipid structure of the
intercellular space may, at least locally and transiently, express two-periodic
curvature (i.e., periodic “bumps” occurring in the lamellar membrane) [45].
2.8
Comparison with Earlier Models
The profound difference between the Landmann model and the membrane-
folding model lies in that the Landmann model includes changes in membrane
topology, while according to the membrane-folding model, topology is kept
constant during barrier formation.
The main advantages of the membrane-folding model with respect to the
Landmann model are the following:
1. Smaller energy cost (involves no budding or fusion)

2. Conserves membrane continuity (preserves water compartmentalization and
allows it to be controlled, since membrane continuity is essential for barrier
function)
3. Allows meticulous control (the thermodynamics of the unfolding procedure
are related to curvature energy)
4. Faster (milliseconds, since membrane unfolding basically represents a phase
transition from cubiclike to lamellar morphology; involves no budding or
fusion)
5. Membrane folding between lamellar and cubiclike morphologies present in
numerous biological systems [[27]; [28], pp. 257–338]
6. Experimental evidence for an “extensive intracellular tubulo-reticular
cisternal membrane system within the apical cytosol of the outermost
stratum granulosum” [18]
7. May explain the reported plethora of forms, numbers, sizes, and general
appearances of “lamellar bodies” in TEM micrographs [13,13]
154 NORLÉN
8. May partly explain the heterogeneous chain length distributions of

epidermal lipids, through a stabilization of three-dimensional hyperbolic
interfaces (showing variations in Gaussian curvature)*
2.9
Proposed Future Experiments
Finally, it is stressed that the alternative barrier-forming process outlined here
relies on the assumption that the true morphology of the lamellar bodies is
different from the conventional notion of discrete bodies. However, preparations
of lamellar bodies isolated by cell fractionation techniques are claimed to
comprise discrete organelles [13, 84, 85]. Nevertheless, the concept of barrier
formation taking place via the folding and unfolding of a single, continuous,
symmetrical three-dimensional structure is intriguing and merits further
experimental examination. One possible way to test the hypothesized continuity
of the proposed barrier-forming three-dimensional structure with symmetry, as
well as the claimed “discreteness” of the lamellar bodies of the Landmann
model, could be by thick-section, high-voltage transmission electron tomography
[2].
It should be noted that, since cubic membranes commonly appear as
multimembranous foldings with cubiclike symmetry, it is not certain that the
proposed continuity of the trans-Golgi network and “lamellar bodies” of the
uppermost stratum granulosum cells as well as the multilamellar lipid matrix of
the intercellular space at the border zone between stratum granulosum and
stratum corneum could be established straightforwardly by, for example, a
molecular tracer study. However, the possibility remains.
3
SKIN BARRIER STRUCTURE AND FUNCTION
A gel phase is usually defined as a crystalline lamellar lipid structure that has
hexagonal hydrocarbon chain packing with rotational disorder along the lipid
chain axes and contains some water between the lamellae ([51], pp. 13– 14, 27;
[86], pp. 16–18, 24–31; [87], pp. 98–101). However, the presence of cholesterol
in a crystalline membrane structure will “solve” the hydrocarbon chains in
*The molecules can then redistribute themselves over regions with variations in Gaussian
curvature and therefore stabilize cubiclike interfaces. This is exemplified by the
observation that a reduction in the monodispersity of self-assembled copolymer systems
may significantly enhance the strength of these materials. Further, admixing polydisperse
homopolymers to lamellar copolymer systems leads to the formation of bicontinuous
hyperbolic mesophases [83].
cholesterol-enriched regions, thereby “melting” the crystalline lipid chain

packing in these areas (from which, consequently, no crystalline wide-angle
reflections can be obtained by means of X-ray diffraction). Therefore, given the
high cholesterol content of the skin barrier lipid matrix (~45 mol % including
cholesteryl esters: Table 1), a “gel” phase is defined here as an extremely tightly
packed liquid crystal* which under certain circumstances may express true
crystalline hydrocarbon chain packing in local cholesterol-deficient regions.
The principal objective of the skin barrier is to be as tight as possible, except
for a minute “leakage” of water needed for the hydration of the keratin of the
corneocytes [88, 89]. The skin must also ensure that the barrier capacity is
optimal even under widely and abruptly changing environmental conditions
(e.g., temperature, pH, salt concentrations, relative humidity, etc.). These
requirements have at least two consequences. (1) sudden transitions of the
physical state of the intercellular lipid matrix of stratum corneum, with possible
different permeabilities between the two phases on either side of the transition
zone and with possible further increased permeability over the whole transition
region due to increased density fluctuations in the membranes [68, 77, 90], and
(2) phase separation between lipids, where permeabilities could be locally
enhanced at the interface between different domains [70, 91–93], will therefore
be avoided as much as possible by the mammalian skin. Thus, from a functional
point of view the stratum corneum intercellular lipid matrix should be as
homogeneous as possible (i.e., ideal physical state, no abrupt phase transitions,
as little phase separation as possible). This can, however, be achieved only by
heterogeneity in the lipid composition, which broadens phase transition zones,
stabilizes gel phases, and ensures that the lamellar morphology remains intact so
that no “pores” or nonlamellar structures are induced [3].
The most characteristic features of stratum corneum lipid composition [8, 11]
are extensive compositional heterogeneity, almost complete dominance of
saturated, very long hydrocarbon chains (20C–36C), and large relative amounts
of cholesterol (~35 mol %: Table 1). It is noteworthy that the stability of “gel”
phases increases with chain length, with compositional impurities (e.g.,
heterogeneity in chain length distributions), and with the presence of cholesterol
[[51], pp. 27, 43–56, p. 412; [94]]. Specifically, the combination of high
cholesterol content and saturated long hydrocarbon chains favors the dissipation
of domain line boundaries [81]. Further, intermixing of different lipid species
may be facilitated by, respectively, the quite stable alkyl chain length
distributions of stratum corneum ceramides and free fatty acids (mainly C24:0–
C26:0) and by the large relative amounts of cholesterol (cf. line active properties
of cholesterol) [95].
*That is, a liquid ordered structure with limited or no lateral diffusion of lipid
hydrocarbon chains.
156 NORLÉN
3.1
The Single Gel Phase Model
Recently a novel model has been published that proposes that the skin barrier (i.e.,
the intercellular lipid within the stratum corneum) exists as a single and coherent
lamellar “gel” phase [3, 4] stabilized by cholesterol and by heterogeneities in
lipid composition (i.e., a liquid ordered structure, or equivalently an extremely
tightly packed liquid crystal, with limited or no lateral diffusion of lipid
hydrocarbon chains, which, under certain circumstances, may express true
crystalline hydrocarbon chain organization in local cholesterol-deficient
regions).
A single and coherent “gel” phase may, in a biological context, be ideal as a
barrier toward the environment. This is because it is a continuous lipid structure
with pronounced compositional heterogeneity that may, irrespective of
environmental (physiologically relevant) conditions, possess low permeability
(owing to the close packing of the saturated long hydrocarbon chains), while
simultaneously being mechanically resistant (owing to its “plasticity” or
“pliability” rendered by the retained rotational disorder of the hydrocarbon
chains in the hexagonal arrangement and/or cholesterol enriched “condensed”
liquid crystal), and expressing little or no tendency for phase transitions, phase
separation, or induction of “pores” or nonlamel lar structures.
3.2
Role of Cholesterol
Cholesterol may be a key component for stratum corneum barrier capacity, since
in vitro it produces the following effects: (1) promotes lamellar structures at high
concentrations ( > 30 mol %) [94] (i.e., decreases the risk for induction of
“pores” or nonlamellar phases with possible harmful effects on barrier function),
(2) increases the chain mobility of lipids in the “gel” state [96] (i.e., renders gel
phases less viscous (or more “plastic”, i.e., “melts” gel phases) and therefore
more mechanically resistant), (3) broadens phase transition regions [94], and
may in some cases entirely abolish subtransitions between gel phases (i.e.,
stabilizes “gel” phases) [81, 97], and (4) probably has line active properties (cf.
two-dimensional surfactant —“lineactant”) in biological membranes (i.e.,
dissipates domain boundaries between domains of different lipid species) [95, 98, 99
]. Consequently, the rigid cholesterol molecular skeleton has the dual effect of
liquefying crystalline and “condensing” liquid crystalline hydrocarbon chains. In
the presence of cholesterol, the movements of liquid crystalline lipid chains are
strongly reduced, with resulting diminished distances between the hydrocarbon
chains and thus increased van der Waals interaction. Consequently, liquid
hydrocarbon chains seem to be “condensed” toward the cholesterol skeleton,
however, without crystallizing and without losing all mobility. For saturated,
crystallized hydrocarbon chains, competition arises for the hydrocarbon chains
between cholesterol and the crystalline aggregate. Cholesterol may consequently

“steal” hydrocarbon chains from the crystalline aggregate by offering these a
more favorable van der Waals interaction (personal communication from Prof.
Irmin Pascher). In such an aggregate with cholesterol, the saturated hydrocarbon
chains cannot be in the all-trans conformation and are thus, by definition, liquid
crystalline (i.e., they cannot give rise to crystalline wide-angle reflections).
Too much cholesterol (above its solubility) will lead to the formation of pure
domains of crystalline cholesterol, and hence will increase the risk
for discontinuities in the lamellar structure. Consequently, for optimum barrier
capacity, the relative amount of cholesterol should be as large as possible but not
above the solubility of cholesterol in the lamellar structure (i.e., ~30 mol %)
[100–103]. In fact, a depressant effect on water permeability (i.e., improved
barrier function) has been observed upon addition of cholesterol to ceramide
containing sphingomyelin and phosphatidylcholine membranes [[71, 101, 104–106
], p. 93]. Further, a high ratio of cholesterol to ceramide has been shown to render
the lamellar lipid organization of mixtures of stratum corneum lipids less
sensitive to variations in skin ceramide composition [107]. Cholesterol thus seems
to stabilize the preferred morphology of the stratum corneum intercellular lipid,
which is in accordance with the single “gel” phase model [3].
3.3
Proposed Molecular Packing Arrangement
The details of the lamellar arrangement of the stratum corneum intercellular lipid
matrix remain highly speculative. In general, a bilayer conformation with
alternating layers of water and lipid is probable. This is because during the
preliminary stages of skin barrier morphogenesis, the ceramides (or their
precursors, the glycosylceramides) should be in a hairpin conformation* (i.e.,
with the two alkyl chains pointing in the same direction) [109], since this is a
prerequisite for them to be solved in a liquid crystalline membrane (cf.
membrane-folding model). During the formation of the crystalline stratum
corneum intercellular lipid matrix, however, a morphological transition of the
ceramides from hairpin to splayed-chain conformation (i.e., with the two alkyl
chains pointing in opposite directions) [109] is possible. This is because the
difference in length between the hydrocarbon chain of the amino alcohol (usually
an 18C sphingosine or phytosphingosine base) and the saturated very long
(usually 24–26C) amide-linked fatty acid of the skin ceramides implies that their
crystal forms pack in a splayed-chain conformation in which the sphingosine and
fatty acid chains form separate matrices [110]. However, in a compositionally
heterogeneous system like the stratum corneum intercellular lipid matrix, which
*The crystal form of a plasma membrane galactosylceramide has been shown to form a
bilayer structure with tilted chains [108].
158 NORLÉN
in addition may contain water, it is not unlikely that the amino alcohols of the
ceramides may be incorporated in the same layers as the amide-linked saturated
long chain fatty acids (i.e., that the ceramides pack in a hairpin conformation)
since both alkyl chains are part of the same molecule. However, as mentioned
earlier, chain bending is encouraged in the C5—C6 bond in conjunction with the
sp2-hybridized C5, and it is manifested in a larger tendency for sphingosine-
based cera mides than for the corresponding phytosphingosine- and
dihydrosphingosine-based ceramides to pack in the hairpin conformation.
It has not yet been established whether water is present between the lamellae of
the stratum corneum intercellular lipid matrix. The possible presence of water,
like the two possible crystalline ceramide conformations (i.e., hairpin and
splayed chain), complicates the interpretation of long spacings reported from
SAXD experiments (64 and 134 Å respectively) [16] and alternating regions of
broad and narrow electron density observed in TEM micrographs [111],
respectively. If the proposed single and coherent lamellar “gel” structure of the
stratum corneum intercellular space contains water between the lamellae, which
is supported by the finding that hydration of the stratum corneum decreases lipid
transition temperatures [112] and increases lipid disordering [113], it would in
turn necessitate the presence of ceramides with a hairpin conformation. On the
other hand, if the proposed single and coherent lamellar gel structure does not
contain any water, a possibility that is supported by the absence of swelling of
the intercellular lipid matrix upon hydration of the stratum corneum [6, 16], the
unique presence of ceramides with a splayed-chain conformation remains an
alternative. However, in both cases (with and without water) the presence of a
combination of hairpin and splayed-chain ceramides is possible [3].
To complicate even further the interpretation of stratum corneum SAXD long
spacings and stratum corneum TEM electron density distributions, the ceramides
of the intercellular lipid matrix may express interdigitation of the hydrocarbon
chains. In addition, different chain packing, with possible different tilts with
reference to the lamellar plane, may occur in separate matrices of the stacked
lamellar structure. However, all the different possibilities for the detailed
molecular organization of the stratum corneum intercellular lipid matrix
mentioned are compatible with the single “gel” phase model [3].
It is difficult to predict whether cholesterol is homogeneously or
inhomogeneously distributed in two and three dimensions. In cholesterol-rich
regions, the single and coherent “gel” phase, predicted by the single “gel” phase
model, may however be an extremely tightly packed (owing to the high content
of saturated extremely long chain lipids and the “condensing” effect of
cholesterol) liquid crystalline phase. Consequently, the lipid organization of the
skin barrier may resemble that of non ionic detergent-resistant membrane
fragments (DRMs)* isolated from a variety of eukaryotic cells. These, like the skin
barrier lipid matrix, are composed of a mixture of saturated long acyl chain
sphingolipids and cholesterol and, likewise, exist as a liquid ordered structure
[114–118].
3.4
Hexagonal Versus Orthorhombic Chain Packing
The notion of a single and coherent lamellar “gel” phase in the lower stratum
corneum is not inconsistent with the WAXD findings of Bouwstra et al. [6].
Bearing in mind that the isolated stratum corneum was studied as a whole, these
investigators could not exclude phase coexistence (e.g., of lipids with
orthorhombic and hexagonal hydrocarbon chain packing, respectively) in
isolated stratum corneum at physiological temperatures. Earlier Bouwstra et al.
[16] had observed that the 134 Å repeat distance disappeared at full hydration
(60 wt % water) (only the 64 Å repeat distance remained), indicating that the
ordering of the structure is more homogeneous at high water concentrations, that
is, in the lower part of stratum corneum facing the viable epidermis.
More recently Pilgram et al. [9] reported the coexistence of orthorhombic
(dominating) and hexagonal chain packing lattices in the lower part of stratum
corneum. These results, obtained by means of electron diffraction may be
explained by noting that, for a pure ceramide (tetracosanoylphytosphingosine) in
the crystalline state, the phase transition from hexagonal to a more orthorhombic
chain packing has been shown to be reversible and continuous (from 106 °C down
to 21 °C) [110]. This implies that the amide-linked fatty acid chains may express
simultaneously an orthorhombic packing in the upper part (i.e., closest to the
polar head group) and a looser, more hexagonal packing in the lower part (i.e.,
the end of the hydrocarbon chain). Thus, in a multicomponent biological system
like the stratum corneum intercellular lipid matrix, the “impurity,” or
compositional heterogeneity (i.e., mixture of many different chain lengths), of
the single and coherent “gel” structure may remain unperturbed (i.e., no or little
lateral diffusion may take place) during the continuous transition from hexagonal
to orthorhombic chain packing in crystalline (i.e., cholesterol-free) areas.* This
in turn implies that the proposed single and coherent “gel” phase may remain
intact: that is, no true thermodynamic phase separation occurs, even in the case
of almost complete “orthorhombicization” of the molecular chain packing in
cholesteroldeficient crystalline regions. In fact, it is a commonly observed
phenom enon in orthorhombic crystals that the most distal two to six
hydrocarbon chain segments express thermal motions and disordered packing.
The mere presence of orthorhombic chain packing in a multicomponent system
like the skin barrier hints (1) at lipid segregation in the vertical (lamellar) and/or
lateral direction with exclusion of noncocrystallizing lipids, (2) at a very low
hydration level and (3) at a probable monolayer organization with splayed-chain
*DRMs are thought to be derived from so-called membrane “rafts.”

*Note that cholesterol may be inhomogeneously distributed in the single and coherent “gel”
phase. Consequently, only cholesterol-free zones (i.e., “microcrystals”) of the proposed
single and coherent “gel” phase can express hexagonal and/or orthorhombic chain
packing.
160 NORLÉN
conformation, of at least phytosphingosinebased ceramide fractions. However,

whether the reported orthorhombic organization of the stratum corneum
intercellular lipid matrix reflects the endogeneous in vivo situation* or
predominantly is a consequence of in vitro tissue preparation and storage† (heat
separation of stratum corneum from epidermis, dehydration in desiccation
chamber, congelation without vitrification of tissue water) are matters that
remain to be elucidated.
3.5
The Desquamation Process
The intact (i.e., ideal or non perturbed, “gel” phase) is perhaps mainly located to
the lower stratum corneum where the water concentration is high, ~40% (w/w)
[58, 59], promoting a higher degree of lipid chain disorder [113]. However,
phase separation, for example, between different crystalline or even liquid
crystalline structures (cf. domain mosaic model of Forslind, Ref. [119]) is likely
to occur in the stratum corneum as a whole, but may predominantly be located to
the upper layers [3]. This is because in the upper part of stratum corneum the
water content is lower (promoting lipid phase transitions and phase separation
[[55, 56], pp. 440–443; 120]), and the heterogeneity of the lipid “fauna” is larger
than in the lower stratum corneum (e.g., cholesterol concentrations may be
suboptimal and medium-chain and unsaturated free fatty acids may be introduced
as a result of breakdown processes and recycling and intermixing with sebaceous
gland lipids during the desquamation process [11, 121].
Since the water content in the stratum corneum decreases closer to the skin
surface, the first lipids to segregate into separate crystals should be those with the
longest, saturated alkyl chains [3]. However, high cholesterol levels may partly
inhibit crystalline segregation (cf. line active properties, Ref. [95]). In addition,
compositional impurities (e.g., broad alkyl chain length distributions) stabilizes
gel phases of for example, alcohols and monoglycerides [[51], p. 27], which also
may be true for the intercellular lipid matrix of the stratum corneum.
*For example, decreased head group polarity and hydratization level rendered the
ceramides after splitting of the glycosyl moieties and the drop in water chemical potential,
at the stratum granulosum-stratum corneum interface.
†For fats, the transition from hexagonal to orthorhombic packing is not reversible (the
melting point of orthorhombic forms being higher than that of corresponding hexagonal
forms) [[86], pp. 40–43; [51], pp. 27–32; personal communication from Prof. Kåre
Larsson and Dr. Lars Hernqvist], which may also be the case for a subfraction of the
relatively nonpolar skin barrier lipids.
3.6
Mechanical Properties
The postulated single and coherent lamellar “gel” phase may very well be
“plastic” enough to resist mechanical stress imposed on the skin. This is
especially true because the lateral expansion of the skin is limited by the form
stability of corneocytes which is internally reinforced by the organization of
anchoring keratin filaments in the plane of the cell [123, 123], and by the
persistence of the desmosomes on the corneocyte cell edges into the final
desquamation [124]. In addition, the macroscale bending of the skin will have a
limited effect on the single lipid molecules on a molecular scale (in the order of
10–5 angular degree per molecule) [3].
3.7
Proteins in the Stratum Corneum Intercellular Space?
A consequence of the proposed single and coherent lamellar “gel” phase of the
stratum corneum intercellular space is that proteins will have very limited
solubility in the stacked lipid layers [[56], p. 270]. However, this does not
exclude that proteins may be associated with the lipid lamellae of the proposed
“gel” structure [3]. Further, complicated lipid-protein interactions may play
important roles in the organization of the skin barrier. However, from a
biophysical perspective, this field remains today almost completely unexplored.
3.8
Comparison with Earlier Models
The first model of the mammalian skin barrier, the brick and-mortar model,
presented by Michaels et al. in 1975, treats the skin barrier as a simplified two-
compartment system with a discontinuous protein compartment embedded in a
continuous, homogeneous lipid matrix [125]. However, the authors pointed out
that both compartments had to be largely heterogeneous. Therefore, the single
“gel” phase model contains no contradictions with respect to the brick and-mortar
model [3].
The basic idea of the present model for the detailed structure of the skin
barrier, the domain mosaic model, presented by Forslind in the mid-1990s [119],
is the presence of coexisting crystalline and liquid crystalline lipid phases (i.e.,
with melted hydrocarbon chains and practically unhin dered diffusivity in the
lateral dimension)[[51], p. 47; 87, p. 49; 126]. However, the following facts
make the presence of a liquid crystalline lipid structure in the stratum corneum
unlikely [3]:
1. Water permeabilities of biological membranes in the gel state and the

intercellular lipid matrix of isolated stratum corneum are in the same order of
162 NORLÉN
magnitude,* that is, approximately two to three orders of magnitude lower

than for liquid crystalline membranes [[127]; personal communication with
Prof. Håkan Wennerström],
2. No endogenous lipids of medium chain length (C12-C18) or with
unsaturated alkyl chains have been detected in the inner/lower stratum
corneum [11] (the hydrocarbon chain length distributions of ceramides and
free fatty acids being quite stable around C24:0-C26:0) [7, 11],
3. The high relative amount of cholesterol (~30 mo1 %), which would prefer to
be associated with medium-chain lipids when these are present [95, 102] and
also has the effect of decreasing molecular chain mobility and mean head
group area of lamellar liquid crystalline structures (i.e., cholesterol makes
these more “gel” -like) [96],
4. The absence of any signs of swelling of the intercellular lipid matrix on
hydration of the stratum corneum [16] (since liquid crystalline phases swell
more readily than gel phases).
Swartzendruber et al. in 1989 [111] and Bouwstra et al. in 1998 [133] presented
detailed models for the molecular organization of the skin barrier based on the
long spacings of SAXD (64 and 134 Å respectively) and the regions of broad and
narrow electron density observed in TEM experiments, respectively. It is
difficult to interpret these results at present, however, because (1) the ceramides
may be in splayed-chain and/or hairpin conformation, (2) water may be present
between the lamellae, (3) hydrocarbon chain interdigitation may occur, and (4)
different chain packing, with possible different tilts with reference to the lamellar
plane, may be present in different matrices of the stacked lamellar structure [3].
The single “gel” phase model differs in a most significant way from earlier
models in that it clearly states that in the unperturbed barrier structure, there is no
true thermodynamic phase separation [3], neither between liquid crystalline and
crystalline phases (cf. the domain mosaic model of Forslind in Ref. [119]) nor
between different crystalline phases with hexagonal and orthorhombic chain
packing respectively [6, 9].
* The permeability to water of the intercellular lipid matrix of isolated stratum corneum is
of the same order of magnitude as the permeability to water of model membranes in the
gel state (0.5– 2.0×10–5 cm/s) [127]. This is evident from them easured permeability of
humanand pigstratum corneum of 0.4–2.0×10–7 cm/s (1.4×10–7 cm/s [128]; 0.4–2.0×10–7
cm/s [129]; 1.1×10–7 cm/s [130]; 0.7×10–7 cm/s [131]) when it is taken into account that
the intercellular lamellar lipid matrix corresponds approximately to 1 to 10% [132] of the
total stratum corneum thickness and that these lipid lamellae essentially are rate limiting
for water diffusion in a direction vertical to the plane of the skin. In addition, water may
be present between the lamellae of the intercellular lipid matrix, further reducing the
effective diffusional path length for water.
3.9
Proposed Future Experiments
One possible way to test the phase homogeneity of the proposed single and
coherent lamellar “gel” structure versus the co existence of liquid crystalline and
crystalline phases (cf. domain mosaic model) as well as the coexistence of
hexagonal and orthorhombic crystalline phases [6, 9] might be by time-resolved
synchrotron X-ray diffraction on lower stratum corneum collected immediately
after tape stripping in vivo.
4
CONCLUSION
It has been proposed that skin barrier formation may take place via a continuous
process of intersection-free membrane unfolding (i.e., a continuous and highly
dynamic deformation of a single and coherent symmetrical three-dimensional
lipid structure into a flat two-dimensional lipid structure) with a concomitant
“crystallization” of the emerging multilamellar lipid structure representing the
developing skin barrier. Recent experimental evidence speaks in favor of this
theory.
The profound difference between the earlier Landmann model and the
membrane folding model lies in that the Landmann model includes changes in
membrane topology while topology is kept constant during barrier formation
according to the membrane folding model.
The main advantages of the membrane folding model over the Landmann
model are the following: (1) energy cost is smaller (involves no budding or
fusion); (2) membrane continuity is conserved (preserves and allows control of
water compartmentalisation bearing in mind that membrane continuity is
essential for barrier function); (3) meticulous control is possible (the
thermodynamics of the unfolding procedure being related to curvature energy);
(4) it is faster (milliseconds, since membrane unfolding basically represents a
phase transition from cubiclike to lamellar morphology and involves no budding
or fusion); (5) membrane folding between lamellar and cubiclike morphologies
has been identified in numerous biological systems; (6) there is experimental
evidence for an “extensive intracellular tubulo-reticular cisternal membrane
system within the apical cytosol of the outermost stratum granulosum”; (7) it
may explain the reported plethora of forms, numbers, sizes and general
appearances of “lamellar bodies” in TEM micrographs.
It is further proposed that the skin barrier (i.e., the intercellular lipid within the
stratum corneum), exists as a single and coherent lamellar “gel” phase (i.e., an
extremely tightly packed liquid crystal, which under certain circumstances may
express true crystalline hydrocarbon chain packing in local cholesterol-deficient
regions). This membrane structure is stabilized by the very particular lipid
composition and lipid chain length distributions of the stratum corneum
164 NORLÉN
intercellular space and has virtually no phase boundaries. The intact (i.e.,
unperturbed, single, and coherent) lamellar “gel” phase is proposed to be mainly
located to the lower half of stratum corneum. Further up (i.e., closer to the skin
surface), crystalline segregation and phase separation may occur as a result of
desquamation.
The single “gel” phase model differs most significantly from earlier models in
that it predicts that no true thermodynamic phase separation, neither between
liquid crystalline and crystalline phases nor between different crystalline phases
with hexagonal and orthorhombic chain packing respectively, is present in the
unperturbed barrier structure.
Further, the proposed single and coherent lamellar “gel” phase does not
necessarily have to contain water, does not necessarily have to be in a bilayer
conformation, and may express an inhomogeneous distribution of lipid
components (e.g., cholesterol).
The main advantages of the single “gel” phase model with respect to earlier
models are that it predicts a continuous barrier structure (i.e., no one- or two-
dimensional phase separation) which is highly impermeable as well as
mechanically resistant (owing to its very particular and heterogeneous lipid
composition) and will remain so, since it does not allow sudden phase transitions,
phase separation, or induction of “pores” or nonlamellar structures, no matter what
the (physiologically relevant) environmental conditions are.
The single “gel” phase model is in accordance with stratum corneum data on
water permeability, lipid compositional as well as SAXD results (absence of
swelling of the intercellular lipid matrix upon hydration). Also, it does not
contradict what has been suggested about skin barrier formation by the
membrane-folding model.
ACKNOWLEDGMENTS
The present work was made possible by the generous support from the Wenner-
Gren Foundations.
REFERENCES
1. Peacocke, AR (1983) An Introduction to the Physical Chemistry of Biological
Organization. Oxford University Press, New York.
2. Norlén, L (2001) Skin barrier formation: the membrane folding model. J. Invest.
Dermatol. 117(4): 823–829.
3. Norlén, L (2001) Skin barrier structure and function: the single gel-phase model. J.
4. Norlén, L (2002) Does the single gel phase exist in stratum corneum? Reply. J.
5. Garson, J-C. Doucet, J. Lévêque, J-L. Tsoucaris, G (1991) Oriented structure in

human stratum corneum revealed by x-ray diffraction. J. Invest. Dermatol. 96(1):
43–49.
6. Bouwstra, JA.Gooris, GS.Salmon-de Vries, MA.Van der Spek, JA. Bras, W (1992)
Structure of human stratum corneum as a function of temperature and hydration: a
wide-angle X-ray diffraction study. Int. J.Pharm. 84 : 205–216.
7. Wertz, PW.Swartzendruber, DC.Kathi, CM. Downing, DT (1987) Composition and
morphology of epidermal cyst lipids. J. Invest. Dermatol. 89 :419– 424.
8. Norlén, L.Nicander, I.Lundh-Rozell, B.Ollmar, S.Forslind, B. (1999) Inter and
intra individual differences in human stratum corneum lipid content related to
physical parameters of skin barrier function in-vivo. J. Invest. Dermatol. 112(1):72–
77.
9. Pilgram, GSK. Engelsma-van Pelt, AM. Bouwstra, JA. Koerten, HK (1999)
Electron diffraction provides new information on human stratum corneum lipid
organization studied in relation to depth and temperature. J. Invest. Dermatol. 113
(3):101–107.
10. Wertz, PW.Norlén, LPO (2002) “Confidence intervals” for the “true” lipid
composition of the human skin barrier? Chapter 5, this volume.
11. Norlén, L.Nicander, I.Lundsjö, A.Cronholm, T.Forslind, B. (1998) A new HPLC-
based method for the quantitative analysis of inner stratum corneum lipids with
special reference to the free fatty acid fraction. Arch. Dermatol Res. 290 : 508–516.
12. Brody, I (1989) A light electron microscopy study of normal human stratum
corneum with particular reference to the intercellular space. Uppsala J. Med. Sci.
94 : 29–45.
13. Madison, KC, Sando, GN.Howard, EJ.True, CA.Gilbert, D.Swartzendruber,
DC.Wertz, PW (1998) Lamellar granule biogenesis: a role for ceramide
glycosyltransferase, lysosomal enzyme transport, and the Golgi. J. Invest.
Dermatol. Symp. Proc. 3(2):80–86.
14. Breathnach AS, Goodman T, Stolinski C, Gross M (1973) Freeze fracture
replication of cells of stratum corneum of human epidermis. J.Anat. 114: 65–81.
15. Elias, PM. Friend, DS (1975) The permeability barrier in mammalian
epidermis.J.Cell Biol. 65 : 180–191.
16. Bouwstra, JA.Gooris, GS.Van der Spek, JA.Bras, W (1991) Structural
investigations of human stratum corneum by small-angle X-ray scattering. J.
Invest. Dermatol. 97:1005–1012.
17. Landmann, L (1986) Epidermal permeability barrier: transformation of lamellar
granule-disks into intercellular sheets by a membrane-fusion process, a freeze-
fracture study. J. Invest. Dermatol. 87(2):202–209.
18. Elias, PM. Cullander, C.Mauro, T.Rassner, U.Kömüves. Brown, BE. Menon, GK
(1998) The secretory granular cell: the outermost granular cell as a specialized
secretory cell. J. Invest. Dermatol. Symp. Proc. 3(2): 87–100.
19. Brody, I (1966) Intercellular space in normal human stratum corneum. Nature 209:
472–476.
20. Matoltsy, AG (1966) Membrane coating granules of the epidermis. J. Ultrastruct.
Res. 15:510–515.
21. Lavker, RM (1976) Membrane coating granules: the fate of the discharged
lamellae. J. Ultrastruct. Res. 55:79–86.
166 NORLÉN
22. Odland, GP.Holbrook, K (1981) The lamellar granules of epidermis. Curr. Problems
Dermatol. 9 :29–49.
23. Fartasch, M (1997) Epidermal barrier disorders of the skin. Microsc. Res.
Techniques 38:361–372.
24. Landh, T (1995) From entangled membranes to eclectic morphologies: cubic
membranes as subcellular space organizers. FEBS Lett. 369:13–17.
25. Lindblom, G. Rilfors, L (1989) Cubic phases and isotropic structures formed by
membrane lipids—possible biological relevance. Biochim. Biophys. Acta 988:221–
256.
26. Hazel, JR.Williams, EE (1990) The role of alterations in membrane lipid
composition in enabling physiological adaptation of organisms to their physical
environment. Prog. Lipid Res. 29:167–227.
27. Landh, T (1996) Cubic cell membrane architectures—taking another look at
membrane bound cell spaces. Thesis, Department of Food Technology, Lund
University, Lund, Sweden.
28. Hyde, S.Andersson, S Larsson, K.Blum, Z.Landh, T.Lidin, S.Ninham, BW (1997)
The Language of Shape. The Role of Curvature in Condensed Matter: Physics,
Chemistry and Biology. Elsevier Science, Amsterdam.
29. Helfrich, W (1973) Elastic properties of lipid bilayers: theory and possible
experiments. Z.Naturforsch. 28c:693–703.
30. Fogden, A Hyde, ST.Lundberg, G (1991) Bending energy of surfactant films. J.
Chem. Soc. Faraday. Trans. 87(7):949–955.
31. Funari, SS. Rapp, G (1999) A continuous topological change during phase
transitions in amphiphile/water systems. Proc. Natl. Acad. Sci. USA 96:7756–
7759.
32. Lindblom, G.Larsson, K.Johansson, L.Fontell, K.Forsén, S (1979). The cubic phase
of monoglyceride-water systems. Arguments for a structure based upon lamellar
bilayer units. J.Am. Chem. Soc. 101:5465–5470.
33. Siegel, DP (1993) Energetics of intermediates in membrane fusion: comparison of
stalk and inverted micellar intermediate mechanism. Biophys. J. 65:2124–2140.
34. Markin, VS. Kozlov, MM. Borovjagin, VL (1984) On the theory of membrane
fusion. The stalk mechanism. Gen. Physiol. Biophys. 5 :361–377.
35. Siegel, DP (1999) The modified stalk mechanism of lamellar/inverted phase
transitions and its implications for membrane fusion. Biophys J. 76:291–313.
36. Seifert, U (1993) Curvature-induced lateral phase segregation in two-component
vesicles. Phys. Rev. Lett. 70 : 1335–1338.
37. Lipowski, R (1995) The morphology of lipid membranes. Curr. Opin. Struct. Biol.
5:531–540.
38. Oparka, KJ. Johnson, PC (1978) Endoplasmatic reticulum and crystalline fibrils in
the root protophloem of Nymphoides peltata. Planta 143 :21–27.
39. Pathak, RK. Luskey, KL. Anderson, RGW (1986) Biogenesis of the crystalloid
endoplasmic reticulum in UT-1 cells: evidence that the newly formed
endoplasmatic reticulum emerges from the nuclear envelope. J. Cell Biol. 102:
2158–2168.
40. von Wettstein, D (1959) The formation of plastid structures. Brookhaven Symp.
Biol. 11:138–159.
41. Gunning, BES (1965) The greening process in plastids. 1. The structure of the
prolamellar body. Protoplasma 60:111–130.
42. Deng, Y. Landh, T (1995) The cubic gyroid-based membrane structure of the
chloroplast in Zygnema (Chlorophyceae zygnematales).Zool. Stud. 34:175– 177.
43. Helfrich, W (1989) Hats and saddles in lipid membranes. Liquid Cryst. 5(6):1647–
1658.
44. Jakob, M. Larsson, K. Andersson, S (1997) Lipid bilayer standing wave
conformation in aqueous cubic phases. Z.Kristallogr. 212:5–8.
45. Larsson, K (1997) On periodic curvature and standing wave motions in cell
membranes. J.Chem. Phys. Lipids 88:15–20.
46. Andersson, S. Larsson, K. Larsson, M. Jacob, M (1999) Biomathematics:
Mathematics of Biostructures and Biodynamics. Elsevier, Amsterdam.
47. Luzatti, V. Tardieu, A. Gulik-Krzywicki, T. Rivas, E. Reiss-Husson, F (1968)
Structure of the cubic phases of lipid-water systems. Nature 220:485–488.
48. Larsson, K (1989) Cubic lipid-water phases: structures and biomembrane aspects. J.
Phys. Chem. 93:7304–7314.
49. Henriksson, U. Blackmore, ES. Tiddy, GJT. Söderman, O (1992) Intermediate
liquid crystalline phases in the binary system C16TACI-H2O: an NMR and low-
angle X-ray diffraction study. J. Phys. Chem. 96:3894–3920.
50. Burgoyne, J. Holmes, MC. Tiddy, GJT (1995) An extensive mesh phase liquid
crystal in aqueous mixtures of a long chain nonionic surfactant. J. Phys. Chem. 99:
6054–6063.
51. Larsson, K (1994) Lipids: Molecular Organisation, Physical Functions and
Technical Applications. Oily Press, Dundee, Scotland.
52. Engstrom, S.Lindahl, L.Wallin, R.Engblom, J (1992) A study of polar lipid drug
carrier systems undergoing a thermoreversible lamellar-to-cubic phase transition.
Int. J.Pharm. 98:137–145.
53. Seddon, JM. Cevc, G. Marsh, D (1983) Calorimetric studies of the gel-fluid
transition (L L ) and lamellar-inverted hexagonal (L HII) phase transition in
dialkyl- and diacylphosphatidylethanolamines. Biochemistry 22:1280–1289.
54. Fournier, JB (1996) Non-topological saddle-splay and curvature instabilities from
anisotropic membrane inclusions. Phys. Rev. Lett. 76(23):4436–4439.
55. Guldbrand, L.Jönsson, B.Wennerström, H (1982) Hydration forces and phase
equilibria in the dipalmitoyl phosphatidylcholine-water system. J. Colloid Interface
Sci. 89(2):532–541.
56. Evans, FD.Wennerström, H (1994) The Colloidal Domain: Where Physics,
Chemistry, Biology, and Technology Meet. VCH Publishers, New York.
57. Siegel, DP.Banschbach, J.Alford, D.Ellens, H.Lis, LJ.Quinn, PJ.Yeagel, PL.Bentz,
J (1989) Physiological levels of diacylglycerols in phospholipid membranes induce
membrane fusion and stabilize inverted phases. Biochemistry 28:3703–3709.
58. Warner, RR.Myers, MC.Taylor, DA (1988) Electron probe analysis of human skin:
determination of the water concentration profile. J. Invest. Dermatol. 90:218–224.
59. von Zglinicki, T.Lindberg, M.Roomans, GM.Forslind, B (1993) Water and ion-
distribution profiles in human skin. Acta Dermatol Venereol. (Stockh.) 73:340–343.
60. Gray, GM. Yardley, HJ (1975) Lipid composition of cells isolated from pig, human
and rat epidermis. J. Lipid Res. 16:434–440.
61. Gray GM, White RJ (1978) Glycosphingolipids and ceramides in human and pig
epidermis. J. Invest. Dermatol. 70:336–341.
62. Gray, GM. Yardley, HJ (1975) Different populations of pig epidermal cells:
isolation and lipid composition. J. Lipid Res. 16:441–447.
168 NORLÉN
63. Wertz, PW. Downing, DT.Freinkel, RK.Traczyk, BS (1984) Sphingolipids of the

stratum corneum and lamellar granules of fetal rat epidermis. J. Invest. Dermatol. 83
(3):193–195.
64. Cox, PC. Squire, CA (1986) Variation in lipids in different layers of porcine
epidermis. J. Invest. Dermatol. 87(6):741–744.
65. Freinkel, RK. Traczyk, TN (1985) Lipid composition and acid hydrolase content of
lamellar granules of fetal rat epidermis. J. Invest. Dermatol. 85:295– 298.
66. Holleran, WM.Takagi, Y.Menon, G.Legler, G.Feingold, KR.Elias, PM (1993)
Processing of epidermal glycosylceramides is required for optimal mammalian
cutaneous permeability barrier function. J. Clin. Invest. 91:1656–1664.
67. Holleran, WM.Takagi, Y.Menon, G.Jackson, SM.Lee, JM.Feingold, KR. Elias, PM
(1994) Permeability barrier requirements regulate epidermal -glucocerebrosidase.
J. Lipid Res. 35:905–911.
68. Marcelja, S.Wolfe, J (1979) Properties of bilayer membranes in the phase transition
or phase separation region. Biochim. Biophys Acta 557:24–31.
69. Mouritsen, OG (1983) Studies on the lack of cooperativity in the melting of lipid
bilayers. Biochim. Biophys. Acta 731:217–221.
70. Papahadjopoulos, D.Jacobson, K.Nir, S.Isac, T (1973): Phase transition in
phospholipid vesicles. Fluorescence polarisation and permeability measurements
concerning the effect of temperature and cholesterol. Biochim. Biophys. Acta 311:
330–348.
71. Blok, MC. Van Deenen, LLM. De Gier, J (1977) The effect of cholesterol
incorporation on the temperature dependence of water permeation through
liposomal membranes prepared from phosphatidylcholines. Biochim. Biophys.
Acta 464:509–518.
72. Sakanishi, A.Mitaku, S.Ikegami, A (1979) Stabilising effect of cholesterol on
phosphatidylcholine vesicles observed by ultrasonic velocity measurement.
Biochemistry 18(12):2636–2642.
73. Faure, C.Tranchant, J-F. Duforc, EJ (1998) Interfacial hydration of ceramide in
stratum corneum model membrane measured by 2H-NMR of D2O. J. Chim. Phys.
95:480–486.
74. Bach, D.Sela, B.Miller, IR (1982) Compositional aspects of lipid hydration. Chem.
Phys. Lipids 31:381–394.
75. Bach, D.Miller, IR (1998) Hydration of phospholipid bilayers in the presence and
absence of cholesterol. Biochim. Biophys. Acta 1368:216–224.
76. Dahlén, B.Pascher, I (1972) Molecular arrangements in sphingolipids. Crystal
structure of N-tetracosanoylphytosphingosine. Acta Crystalign B28:2396–2404.
77. Doniach, S (1977) Thermodynamic fluctuations in phospholipid bilayers. J. Chem.
Phys. 68(11):4912–4916.
78. Nagle, JF. Scott, HL (1978) Lateral compressibility of lipid mono- and bi-layers,
theory of membrane permeability. Biochim. Biophys. Acta 513: 236–243.
79. Copeland, BR. McConnell, HM (1980) The rippled structure in bilayer membranes
of phosphatidylcholine and binary mixtures of phosphatidylcholine and
cholesterol. Biochim. Biophys. Acta 599:95–109.
80. Mortensen, K.Pfeiffer, W.Sackmann, E. Knoll, W (1988) Structural properties of a
phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering
—Ripple structure and phase diagram. Biochim. Biophys. Acta 945:221–245.
81. Slotte, JP (1995) Lateral domain heterogeneity in cholesterol/

phosphatidylosphatidylcholine monolayers as a function of cholesterol
concentration and phosphatidylcholine acyl chain length. Biochim. Biophys. Acta
1238: 118–126.
82. Sparr, E.Wennerström, H (2001) Responding phospholipid membranes—interplay
between hydration and permeability. Biophys. J. 81(2):1014–1028.
83. Hasegawa, H.Hashimoto, T.Hyde, ST (1996) Microdomain structures with
hyperbolic interfaces in block and graft polymers. Polymer 37(17): 3825–3833.
84. Freinkel, RK.Traczyk, TN (1981) A method for partial purification of lamellar
granules from fetal rat epidermis. J. Invest. Dermatol. 77:478–482.
85. Grayson, S.Johnson-Winegar, AG.Wintroub, BU.Epstein, EH. Elias, PM (1985)
Lamellar body-enriched fractions from neonatal mice: preparative techniques and
partial characterization. J. Invest. Dermatol. 85: 289–295.
86. Hernquist, L (1984) Polymorphism of fats. Thesis, Department of Food
Technology, Lund University, Lund, Sweden.
87. Small DM (1986) The Physical Chemistry of Lipids. Handbook of Lipid Research.
Plenum Press, New York.
88. Blank IH (1952) Factors which influence the water content of stratum corneum. J.
Invest. Dermatol. 18:433–440.
89. Guyton, AC. Hall, JE (1996) Textbook of medical physiology. WB Suanders,
Philadelphia, p 396.
90. Georgallas, A. MacArthur, JD. Ma, X-P. Nguyen, CV.Palmer, GR.Singer, MA.
Tse, MY (1987) The diffusion of small ions through phospholipid bilayers.
J.Chem. Phys. 86(12):7218–7226.
91. Cruzeiro-Hansson, L.Mouritsen, OG (1988) Passive ion transport of liquid
membranes modelled via lipid-domain interfacial area. Biochim. Biophys. Acta
944:63–72.
92. Clerc, SG.Thompson, TE (1995) Permeability of dimyristoyl phosphatidylcholine/
dipalmitoyl phosphatidylcholine bilayer membranes with coexisting gel and liquid-
crystalline phases. Biophys. J. 68:2333–2341.
93. Xiang, TX.Anderson, BD (1998) Phase structures of binary lipid bilayers as
revealed by permeability of small molecules. Biochim. Biophys. Acta 1370:64–76.
94. Takahashi, H.Sinoda, K.Hatta, I (1996) Effects of cholesterol on the lamellar and
the inverted hexagonal phases of dielaidoylphosphatidylethanolamine. Biochim.
Biophys. Acta 1289:209–216.
95. Sparr, E.Ekelund, K.Engblom, J.Engstrom, S.Wennerström, H (1999) An AFM
study of lipid monolayers: II. The effect of cholesterol on fatty acids. Langmuir 15
(20):6950–6955.
96. de Kruyff, B. van Dijck, PWM. Demel, RA.Schuijff, A.Brants, F. van Deenen,
LLM (1974) Non-random distribution of cholesterol in phosphatidylcholine
bilayers. Biochim. Biophys. Acta 356:1–7.
97. McMullen, TPW. McElhaney, RN (1995) New aspects of the interaction of
cholesterol with dipalmitoyl phosphatidylcholine bilayers as revealed by high-
sensitivity differential scanning calorimetry. Biochim. Biophys. Acta 1234:90–98.
98. Weis, RM. McConnel, HM (1985) Cholesterol stabilizes the crystal-liquid interface
in phospholipid monolayers. J.Phys. Chem. 89: 4453–4459.
170 NORLÉN
99. Cruzeiro-Hansson, L.Ipsen, JH.Mouritsen, OG (1989) Intrinsic molecules in lipid

membranes change the lipid-domain interfacial area: cholesterol at domain
interfaces. Biochim. Biophys. Acta 979:166–176.
100. Subramaniam, S.McConnell, M (1987) Critical mixing in monolayer mixtures of
phospholipid and cholesterol. J.Phys. Chem. 91:1715–1718.
101. Lieckfeldt, R.Villalain, J.Gomez-Fernandez, JC.Lee, G (1993) Diffusivity and
structural polymorphism in some model stratum corneum in some lipid systems.
Biochim. Biophys. Acta 1151:182–188.
102. Engblom, J.Engstrom, S.Jönsson, B (1998) Phase co-existence in cholesterol-fatty
acid mixtures and the effect of the penetration enhancer Azone. J. Control. Rel. 52:
271–280.
103. Norlén, L.Engblom, J (2000) Structure-related aspects on water diffusivity in fatty
acid-soap and skin lipid model systems. J. Controlled Release 63(1-2):213–226.
104. Finkelstein, A.Cass, A (1967) Effect of cholesterol on the water permeability of
thin lipid membranes. Nature 216:717–718.
105. Fettiplace, R (1978) The influence of lipid on the water permeability of artificial
membranes. Biochem. Biophys. Acta 513:1–10.
106. Schaefer, H.Redelmeier, TE (1996) The Skin Barrier—Principles of Percutaneous
Absorption. Karger, Basel.
107. Bouwstra, JA. Dubbelaar, FER. Gooris, GS. Weerheim, AM. Ponec, M (1999) The
role of ceramide composition in the lipid organisation of the skin barrier. Biochim.
Biophys. Acta 1419(2):127–136.
108. Pascher, I.Sundell, S (1977) Molecular arrangements of sphingolipids. The crystal
structure of cerebroside. Chem. Phys. Lipids 20:175–191.
109. Corkery, RW. Hyde, ST (1996) On the swelling of amphiphiles in water. Langmuir
12(23):5528–5529.
110. Dahlén, B. Pascher, I (1979) Molecular arrangements in sphingolipids. Thermotropic
phase behaviour of tetracosanoylphytosphingosine. Chem. Phys. Lipids 24:119–
133.
111. Swartzendruber, DC.Wertz, PW.Kitko, DJ.Madison KC. Downing DT (1989)
Molecular models of the intercellular lipid lamellae in mammalian stratum
corneum. J. Invest. Dermatol. 92 :251–257.
112. Golden, GM.Guzek, DB.Harris, RR.McKie, JE.Potts, RO (1986) Lipid
thermotropic transitions in human stratum corneum. J. Invest. Dermatol. 86:255–
259.
113. Alonso, A.Meirelles, NC. Tabak, M (1995) Effect of hydration upon the fluidity of
intercellular membranes of stratum corneum: an EPR study. Biochim. Biophys.
Acta 1237:6–15.
114. Ahmed, SN. Brown, DA. London, E (1997) On the origin of sphingolipid/
cholesterol-rich detergent-insoluble cell membranes: physiological concentrations
of cholesterol and sphingolipid induce formation of a detergentinsoluble, liquid-
ordered lipid phase in model membranes. Biochemistry 36:10944–10953.
115. Brown, DA. London, E (1997) Structure of detergent-resistant membrane domains:
does phase separation occur in biological membranes? Biochem. Biophys. Res.
Commun. 240:1–7.
116. Brown, RE (1998) Sphingolipid organization in biomembranes: what physical
studies of model membranes reveal. J. Cell Sci. 111:1–9.
117. Ge, M.Field, KA.Aneja, R.Holowka, D.Baird, B.Freed, JH (1999) Electron spin
resonance characterization of liquid ordered phase of detergent-resistant
membranes from RBL-2H3 cells. Biophys. J. 77:925–933.
118. Xu, X. London, E (2000) The effect of sterol structure on membrane lipid domains
reveals how cholesterol can induce lipid domain formation. Bio-chemistry 39(5):
843–849.
119. Forslind B (1994) A domain mosaic model of the skin barrier. Acta Dermatol
Venereol. (Stockh.) 74 :1–6.
120. Sparr, E. Wennerström, H (2000) Diffusion through a lamellar liquid crystal: a
model of molecular transport across stratum corneum. Colloids Surf. B:
Biointerfaces 19:103–116.
121. Bonté, F. Saunois, A. Pinguet, P. Meybeck, A (1997) Existence of a lipid gradient
in the upper stratum corneum and its possible biological significance. Arch.
Dermatol Res. 289:78–82.
122. Swanbeck, G (1959b) Macromolecular organisation of epidermal keratin. Acta
Dermatol Venereol. (Stockh.) (suppl. 43) 39:1–37.
123. Norlén, L. Emilson, A. Forslind, B. (1997) Stratum corneum swelling. Bio-
physical and computer assisted quantitative assessments. Arch. Dermatol Res. 289 :
506–513.
124. Mils, V.Vincent, C.Croute, F.Serre, G (1992) The expression of desmosomal and
corneodesmosomal antigens shows specific variations during terminal
differentiation of epidermis and hair follicle epithelia. J Histochem. Cytochem. 40:
1329–1337.
125. Michaels, AS. Chandrasekaran, SK.Shaw, JE (1975) Drug permeation through
human skin: theory and in vitro experimental measurements. AIChE J. 21 (5):985–
996.
126. Friedel, MG (1922) Les états mésomorphes de la matière. Ann. Phys. (Nov.-Dec.)
pp. 273–474.
127. Carruthers, A. Melchior, DL (1983) Studies of the relationship between bilayer
water permeability and bilayer physical state. Biochemistry 22: 5797–5807.
128. Scheuplein, RJ. Ross, L (1970) Effects of surfactants and solvents on the
permeability of epidermis. J. Soc. Cosmet. Chem. 21:853–873.
129. Blank, IH. Moloney, J.Emslie, AG.Simon, I. Apt, C (1984) The diffusion of water
across the stratum corneum as a function of its water content. J. Invest. Dermatol.
82(2):188–194.
130. Potts, RO. Francoeur, ML (1991) The influence of stratum corneum morphology
on water permeability. J. Invest. Dermatol. 96(4):495–499.
131. Edwards, DA. Langer, R (1994) A linear theory of transdermal transport
phenomena. J Pharm. Sci. 83(9):1315–1329.
132. Bouwstra, JA. Gooris, GS. Dubbelbelaar, FER. Weerheim, AM. Ijzerman, AP.
Ponec, M (1998) Role of ceramide 1 in the molecular organisation of the stratum
corneum lipids. J. Lipid Res. 39:186–196.
8
The Skin Barrier: An Evolutionary and
Environmental Perspective
Gopinathan K.Menon
California Academy of Sciences, San Francisco, California, U.S.A
In an unusual scenario where religion imitates evolution, the various incarnations

of the Hindu God represent sequentially the higher forms of vertebrates—
beginning with the fish, a turtle, a beast-man, and then human forms—
evolutionary steps most obvious in the morphological appearance and
specializations of the integument. Notwithstanding the ideal separation of church
from the “state of science,” one could venture to say that all skins may not be
created equal, but all have evolved to suit perfectly the environmental needs of
specialized habitats. Today when Homo sapiens are “creating” skin or skin
equivalents via tissue engineering for use in medical and research applications,
appreciation of an evolutionary and environmental perspective of the primary,
secondary, and even tertiary barrier functions of skin could be extremely useful.
As stated ad nauseam, the skin is the largest and perhaps the most complex organ
system, and its multitudinous true functions continue to stimulate discussions in
interdisciplinary scientific forums [1]. The primary function of skin, as
commonly agreed, is protection: that is, it functions as the body’s primary
defense—a barrier to water loss (preventing death by desiccation) or water influx.
Depending on the challenges of the habitat, the barrier function also extends to
ice crystal propagation, ultraviolet (UV) radiation, xenobiotics, microbial
pathogens, oxidants, biofouling, defense from predators by camouflage or
chemical secretions, and so on, as will be discussed in sections to follow. A good
measure of the skin’s barrier properties, at least in the terrestrial species, is
transepidermal water loss (TEWL). Most factors that affect the barrier properties
(such as mechanical stripping of the outermost layers, selective removal of
lipids, or acute UV insult) affect TEWL, as well as setting in motion a process of
barrier repair or barrier homeostasis. The integumentary response to barrier
disruption (or challenge) tends to holistic nature: not selectively upregulating a
specific defense, but rather fortifying the entire gamut of barriers. This is
manifested when tape stripping of stratum corneum not only leads to restoration
of the permeability barrier, but also stimulates melanogenesis (a barrier against
UV) and often hyperpigmentation in darker phototypes. The relationship
between UVand human skin pigmentation is also considered to be an adaptation
that protects sweat glands from UV-induced injury, ensuring the integrity of
174 MENON
somatic thermoregulation, as well as preventing folate deficiency and hence birth

defects [2]—a function that is of obvious value to the species.
1
SKIN AS A BARRIER TO PHYSICAL FORCES:
IMPACT RESISTANCE
Because skin of active vertebrates needs to be flexible, the rigid exoskeleton of
invertebrates had to be replaced over the course of evolution by a tough, yet
somewhat pliable sheath. This modification has taken several forms: the
overlapping scales of fish and reptilians (the hard scales of crocodilians and the
carapace of turtles included), feathers in birds, and fur in mammals. Extreme
specializations in the epidermal appendages such as the quills of hedgehogs and
porcupines, as well as the armor of scaly anteaters and armadillos, also serve this
purpose.
In relatively hairless species such as the rhinoceros, as well as in aquatic
mammals such as the hippopotamus (amphibious) and whales and dolphins
(marine), the loss of such epidermal appendages is adequately compensated by
thickened and specialized dermis and/or subcutaneous adipose tissue. The
thickness and mechanical properties of skin show sitespecific variations; but in
large herbivores, such specializations in specific zones correlate with a shielding
function (e.g., against blows received during intraspecific combat) and with
fighting styles and weapons (horns, teeth, etc.) used [3]. Shadwick et al. [4] who
investigated the structure and mechanical design of the skin of the rhinoceros,
correlated the highly cross-linked collagen fibers (relatively high tensile
strength, elastic modulus, and work of fracture, compared with other mammals)
in the back and flank and found a structural organization that provides high
resistance to compressive failure and tearing by a horn in combat. In the
hippopotamus, the skin is comparatively thin in the belly, but very thick (15–
20mm), stiff, and relatively inextensible along the back, flank, and rump,
minimizing the risk of wounding in intraspecific combat. Similar adaptations are
also seen in walrus skin. As opposed to species that indulge in actual combat, the
skin of elephants (which use threatening displays, rather than actual combat, to
assert dominance) does not function as armor.
The stratum corneum (SC) by itself provides for limited impact resistance by
the “bricks and mortar” arrangement of corneocytes and intercellular lipids,
which makes it a composite biopolymer [5]. Stress propagation along the keratin
filaments, and via desmosomes to adjacent corneocytes, could be adaptations at
the cellular level. An unusual abundance of desmosomes in the SC of rhinos is
indicative of role in impact resistance, in addition to increased SC cohesion.
Lipid droplets within the “lipokeratinocytes” of cetaceans potentially create a
cushioning effect and aid in small deformations of individual corneocytes to
comply with hydrodynamic forces at the boundary layer. Human SC also offers a
formidable physical barrier to particle-mediated gene delivery, as revealed when
THE SKIN BARRIER 175
many gold particles intended for delivery via a gene gun were shown to be
trapped within the SC [6].
2
PERMEABILITY BARRIER
The permeability barrier is primarily considered to be a barrier to the influx and
efflux of water, which is measured as a function of TEWL in terrestrial forms, by
means of various instrumental approaches including ventilated (Meeco
electrolytic moisture analyzer) and non ventilated (Evaporimeter, TEWA meter,
etc.) systems [7]. Although it is not practical to apply these techniques to aquatic
animals, we may safely assume that they do possess a barrier that is appropriate
for survival in their habitats. Most of the organisms employ lipids as the
“waterproofing” moiety [8], to curtail movement of water across the integument.
In higher vertebrates, morphological studies that identify the epidermal
production and secretion of specialized lipid-enriched organelles (e.g.,
membrane-coating granules, mesos granules, multi-granular bodies, Odland
bodies, lamellar bodies) and sequestration of lipids in the SC extra-cellular
domains have provided evidence of barrier formation [9]. A minimalistic
description of the sequence of events in barrier formation in a typical mammal is
as follows. As the basal keratinocytes proliferate and move up or outward into
the stratum spinosum, they embark on a path of progressive differentiation that
involves synthesis of specific proteins (keratins, filaggrin, cornified envelope
proteins, etc.) and lipids (in the form of lamellar granules). In the outermost cells
of the stratum granulosum (2–3 cells thick), about 20% of the cytosol is occupied
by lamellar bodies, which remain interconnected by a trans-Golgi-like membrane
system [10], the rest of the cytosol being predominantly occupied by
keratohyalin granules, keratin filaments, ribosomes, and mitochondria.
When appropriate cellular signals are received, the granulocyte (1) begins to
assemble a cornified envelope from cross-linking of the envelope proteins
(involucrin, loricirn, cornifin), catalyzed by the enzyme transglutaminase (2)
secretes the lamellar bodies into the stratum granulosumstratum corneum
interface, and (3) transforms into a transitional cell (displaying extensive
dissolution of the nuclei and cytosolic organelles) that matures into a terminally
differentiated corneocyte within a few hours [11]. Upon secretion, the disk
contents of individual lamellar bodies (polar lipids) unfurl, fuse end to end with
each other, anchor onto the desmosomes, and by a series of metabolic processing
events mediated by a battery of enzymes (cosecreted with the lipids) are
converted to multiple lamellar structures composed of nonpolar barrier lipids
that fill the intercellular domains of the stratum corneum, imparting a “brick and
mortar” organization to this tissue. These extracellular lipids occluding the
tortuous extracellular space of the corneocytes are the physical location of the
permeability barrier. Further evidence that they are indeed the location of the
permeability barrier is obtained from electron microscopy, showing that
176 MENON
subcutaneously injected, water-soluble tracers (such as lanthanum) moves

outward through the intercellular domains of nucleated epidermis, but its efflux
is stopped where the secreted contents of lamellar bodies occlude the interface
between stratum corneum and stratum granulosum (SG) [12].
The permeability barrier resides in similar locations in submammalian species
of vertebrates as well, providing protection from ice crystal propagation in
hatchling turtles [13] and possibly fishes, where mucoid materials rather than
lipids are produced by epidermis. The organization of highly hydrophobic barrier
lipids in mammals is susceptible to damage by prolonged hydration. Recent
findings of Warner and colleagues [14] on such damage include “roll-up” of the
lamellae and extensive vesiculation in the SC extracellular domains. This
observation leads one to believe that appropriate adaptive features for constant
hydration should have evolved in the skin of marine mammals as their terrestrial
ancestors took to an aquatic mode of life. Comparative studies on vertebrate
integument also show that all barriers are not based on lamellar bodies and that
glandular secretions akin to sebum are also significant factors in the permeability
barrier of many species.
3
THE WATER-TO-LAND TRANSITION
As evolution of amphibians occupies a phylogenic space between fishes and
terrestrial reptiles [15], members of this class of vertebrates offer valuable
insights into integumentary adaptations for this transition. Amphibians with a
free-living, fully aquatic postembryonic (tadpole) stage in their life history
exemplify how ontogeny repeats phylogeny. As the fishlike tadpole undergoes
metamorphosis involving extensive tissue remodeling, organogenesis, and
programmed cell death, complex changes occur in the skin within a short time. The
larva-specific cells (Fig. 1) are replaced by adult keratinocytes (Fig. 2), dermis
and dermal glands develop, and an integument that is functional in both water
and land emerges. Yet, amphibians are not endowed with a good permeability
barrier: the evaporative water loss from skin is nearly equal to that of a free
water surface in most cases. Because these animals employ cutaneous respiration
(in addition to pulmonary mode) and use integument for water uptake and ion
exchange, amphibians have severe distributional constraints. Consequently, most
species can live only in areas with good rainfall. Adaptations of many of the
amphibian species to maintain water balance are primarily behavioral—via
reduced activity and use of favorable microhabitats. However, certain species of
tree frogs have evolved mechanisms to reduce cutaneous water loss to low
levels, although not to levels comparable to that of reptiles. Lillywhite and Mittal
[15] have reviewed the various mechanisms suggested to be responsible for
decreasingTEWL in amphibians, such as thickness and degree of keratinization,
tight junctions, intercellular or external mucus, ossification, calcified “ground
substance,” iridophores, cocoons, and lipids. They conclude that lipids are indeed
the basis of barrier in the most waterproof amphibians. Cocoons in hibernating

frogs, comprising of 40 to 60 layers of stratum corneum cells with secreted
substances forming a “mortar” impose considerable resistance to passage of
water, allowing the frogs to burrow in shallower depths during drought than are
possible for frogs without cocoons. In the burrowing frog Cyclorana australis,
the cocoons reportedly contain 75 to 85% proteins and 5 to 10% neutral lipids.
The most effective epidermal permeability barriers in amphibians are seen in
certain tree frogs that secrete lipids from specialized glands. The South American
tree frog Phyllomedusa sauvagei has lipid secreting glands on the dorsum the
secretions of which are spread over the skin by a complex “wiping behavior” of
both fore and hind legs [16]. The secretion contains wax esters, triglycerides,
free fatty acids, hydrocarbons, and cholesterol. The adaptive value of this lipid
coating is that it curtails water loss, but as temperatures rise and survival depends
on evaporative cooling, these lipids melt, allowing evaporative cooling—a case
of facultative waterproofing. Wiping behavior has since been recorded for an
Australian hylid frog and an Indian tree frog [15], but in the latter, mucus is
mixed in with lipids, and the frogs are not very waterproof. The African species
Chiromantis xerampelina has exceptionally waterproof skin on the dorsum [17],
but a “leakier” skin on the ventral surface. Although the histology and
ultrastructure of skin have been examined (Drewes and Menon, unpublished), the
basis of its rather exceptional permeability barrier remains enigmatic.
3.1
Terrestrial Adaptations
The evolution of amniotes, which exhibit renewal of epidermis throughout their
life, heralded truly terrestrial adaptations among vertebrates. In birds and
mammals, the loss and replacement of feathers and fur (epidermal appendages)
is quite obvious, but exfoliation from interfollicular epidermis is almost
imperceptible. In contrast, lepidosaurian reptiles show a more dramatic “skin
shedding,” either in large pieces (lizards) or “in toto” (snakes).
3.1.1
Reptiles
Reptiles are the first class of vertebrates that evolved true terrestrial adaptations
(cledoic eggs, amniotic membranes) and exhibit low rates of evaporative water
loss suitable to their environment. In addition, since acclimatization (for about 8
days) of lizards to dry air decreases TEWL, compared with conspecifics
acclimated to humid air it is also that rate of evaporative water loss has a genetic
as well as an adaptive component [18]. This “barrier upregulation” has been
attributed to newly formed lipids deposited within the epidermis. In reptiles such
as turtles and tuataras [19], the epidermis elaborates both mucoid granules and
lipid-enriched mesos granules—possibly related to the semiaquatic life of these
178 MENON
FIGURE 1 Electron micrograph of the epidermis of the tadpole, Rana catesbiana. The
larval cell types are the less osmiophilic skein (SK) cells and the outer layer of apical cells
(AP) that actively secretes mucus (arrows) onto the surface (OsO4 postfixation). (Courtesy
of J.Menon.)
animals. Much of the available information on reptilian skin is on lizards and
FIGURE 2 Outer epidermis of adult frog. Note the keratinization and thickened
membrane and lack of mucus secretion in outermost cornified cell. The gap within the
epidermis (arrow) is an artifact of tissue processing (OsO4 post fixation).
snakes (lepidosaurians), which share many similarities of epidermal organization

180 MENON
and renewal pattern (molting in large pieces in lizards, in toto in snakes). This
discussion on reptilian skin barrier is based on the snake (squamate) model.
Although it is a stratified epithelium like that of other amniotes, lepidosauran
epidermis shows some fundamental differences that make it rather unique. First,
the skin is covered with overlapping scales, with flexible hinge regions between
the scales. The thick cornified tissues on the outer surface makes the scales thick
and inflexible, while the inner scale surface and hinge regions with thinner
cornified tissues permit flexibility and distensibility of the integument, needed for
locomotion and feeding. The relatively loose connection between the deeper
dermis and the muscle fascia further aid in this capacity [20]. Second, cells
derived from the basal keratinocytes do not result in a single type of terminally
differentiated corneocyte; rather, they form a complex “epidermal generation”
comprising six morphologically distinct layers and representing alternating
pathways of (outermost layers) and keratinization (inner layers) as well.
Third, the cell loss from epidermis is not by imperceptible desquamation as in
mammals, but by cyclic or seasonal ecdysis; in snakes, there is a whole-body
shedding of the entire “epidermal generation” that involves a two-cell-layer
shedding complex [20] between an old epidermal generation and a new
generation formed underneath.
As seen in Figure 3, the typical scale shows an outermost oberhautchen,
followed by a layer where cell boundaries are obliterated, forming a
syncytium, filled with -keratin and occasional pigment granules, but lacking
any evidence of lipids. In the inner scale surface and hinge regions, the -keratin
is limited to the oberhautchen alone, permitting flexibility here [21]. Subjacent to
the layer is the mesos layer, -keratinizing cells with mesos granule-derived lipid
bilayers within the extracellular domains. Freeze-fracture and electron
microscopic investigations using tracer permeation have shown that the mesos
layer is indeed the location of the permeability barrier in snakes [22]. The
overlying layer clearly affords physical protection to the crucial but delicate
barrier structures of the mesos layer. Below the mesos layer is the layer,
synthesizing -keratin, as well as lipids. Underneath the layer (and above the
germinal/basal cell layer) may be seen the lacunar layer (the clear layer), or
undifferentiated living cells, depending on the stage of skin shedding (i.e.,
resting, renewal, or shedding phase). The cellular mechanisms involved in the
formation of the epidermal generation, the various stages in the shedding cycle,
and its seasonality and endocrine control are complex, and for the most detailed
accounts, the reader is referred to excellent reviews by Maderson and colleagues
[19, 20].
The structural organization of the permeability barrier in reptiles, especially in
lepidosaurians, raises interest in the mechanism(s) of adaptative fortiflcation of
the barrier without having to renew the whole epidermal generation. More
precisely, is the barrier function limited to the mesos layer, or do secretions from
underlying cells further strengthen the barrier? Studies on “wounding” the
scales by tape stripping show that the “repair” primarily involves hyperplasia of
the a layer, but not the formation of and mesos layers. The decreased TEWL
achieved without of skin shedding by lizards that have become acclimated to dry
air [18] also points to the ability of the epidermis for initiating the lipogenesis
needed for facultative water-proofing. What is not known is whether such
conditions result in deposition of lamellar lipid bilayers within the layers. In
snakes, postnatal ecdysis decreases TEWL to adult values owing to an increase
in cell layers of the mesos layer, as well as improved structural organization of
the lamellar lipids [23]. However, in preshed stages, organelles similar to the
lamellar bodies (mesos granules) are also present in the layer, an indication of
the “reserve barrier mechanism” that exists in this layer. Whether they are
secreted as lamellar sheets or transformed to lipid droplets (as in birds) prior to
secretion, and whether their secretion from the layer contributes to facultative
waterproofing, are questions that need to be resolved.
3.1.2
Mammals
As opposed to squamate reptiles, whose periodic skin shedding leads to a build
up of the permeability barrier through formation of a new epidermal generation,
mammals continuously regenerate of the barrier by differentiation of new
keratinocytes and desquamation from the outermost layers of SC. This mode of
epidermal differentiation may provide superior ability for fine-tuning the barrier.
As the cells of stratum granulosum await signals for terminal differentiation, they
continue to behave as “secretory” cells (Fig. 4), retaining an ability to respond to
barrier disruption [10] with massive secretion of nascent lamellar bodies, as well
as upregulated synthesis and secretion of new lamellar bodies. This ability of
mammalian epidermis may underlie the site-specific as well as large intra- and
interindividual variations in TEWL in humans. Under severe environmental
insults, such as a large dose of UV radiation, the mammalian permeability barrier
repair pattern could be reminiscent of reptilian skin shedding, with a new
epidermal generation occuring under the damaged areas.
3.2
Fossorial Adaptations
While many mammals live in burrows, the one most specialized for a
subterranean life is the naked mole rat (Heterocephalus glaber) from the
semidesert areas of southern Ethiopia, Somalia, and Kenya. These animals live in
large colonies in dark, warm (32–34°C) burrows that are humid, and poorly
ventilated. Naked mole rats have poor thermoregulation; both their resting
metabolic rate and body temperature are lower than these of a typical mammal.
Hence members of the colony keep in close physical contact with each other
most of their lives. The skin is a translucent pink and, most of the melanocytes
are restricted to the dermis as in other poikilothermic animals. However, Tucker
182 MENON
FIGURE 3 Reptilian epidermis: ultrastructure of the snake scale showing the outer
syncytical layer (BL), mesos layer (ML), layer (AL); followed by immature cells
and the stratum basale (SB), resting on the basement membrane (BM). Lipid bilayers
deposited within the mesos layer are not visualized under the OsO4 postfixation employed
here. (Skin sample courtesy of Dr. H.Lillywhite.)
[24] notes that when exposed to sunlight, the mole rat’s skin becomes a little
darker, turning brown after exposure to strong sun for 2 days, an indication that a
FIGURE 4 Mammalian epidermis: ultrastructure of the stratum granulosum (SG) and

parts of SC of murine skin, showing cytosolic lamellar bodies (LBs), as well as secreted
LB contents at the SG-SC interface (OsO4 postfixation). (Inset) Basal cell of human
epidermis showing melanosomes (M) capping the nucleus (N)—the UV barrier to protect
the proliferative component of epidermis.
UV barrier may be upregulated when needed. The dorsal skin lacks sweat glands
and hair follicles. In addition, the skin is exceptionally loose, reducing the
stresses when the mole rats are digging or moving along their narrow tunnels.
184 MENON
Digging adaptations include the lack of an external ear (pinna) and the presence
of a nasolabial sensory patch, which can be partially shielded by a transient
buccal invagination [24]. Adipose tissue is well developed in the dermis.
When fed with vegetables with high water content, these animals reportedly
accumulate large subcutaneous fluid bubbles (some as large as the head), which
persist for a few months, without any adverse effects. This strange feature may
be an indication that they do not have much evaporative water loss while in the
burrows. Histologically, the epidermis is relatively thick, with 8 to12 cell layers,
but the dermal-epidermal junction is flat and devoid of rete pegs [25]. However,
the external surface of epidermis (SC) has several folds and keratinous pegs. The
thickness of stratum granulosum is 1 to 3 layers (variable and often said to be
discontinuous), possibly owing to infoldings of the SC at various regions.
The thickness of the stratum spinosum (SS) also varies. In many ways, the
typical adaptive organization of skin to external stress (friction) exemplified by a
highly undulating dermoepidermal junction in mammals (as seen in
palmoplantar areas) is reversed in the naked mole rat—to create a loose skin with
a highly flexible and undulating external surface, but allow for the movement of
the body “inside” the integument. This is diametrically opposed to the
specialization of cetaceans (extremely developed rete pegs, a very smooth
external surface), where the need for hydrodynamic efficiency dictates surface
sculpturing. Such differences support the contention that skin morphology, is a
better indicator than phylogeny of habitat and lifestyle [26]. The lack of rete pegs
also decreases the blood supply to the periphery and may be related to the
poikilothermic nature of the naked mole rats, which rely more on behavioral
(huddling) thermoregulation. Examination of the permeability barrier lipid
organization and TEWL in this species should be interesting.
3.3
Adaptations in Land-to-Air Transition
The most significant aerial adaptations are reduction in body weight (skeletal
system), bipedality (freeing up of forelimbs to become wings), powered flight
(muscle), and unique epidermal derivatives—the feathers (a
significant adaptation for surface contouring and aerodynamics of flight).
Integumentary adaptations [27, 28] include loose attachment to the muscle layers,
lack of sweat glands and sebaceous units associated with feathers (prevents
clumping of feathers), and specialization of feathers into down (insulation),
filoplume (sensory), contour (surface contouring), and quill (flight) feathers. An
effete stratum corneum with non-bilayer-forming lipids allows high basal TEWL
for evaporative cooling, to compensate for the high basal metabolic rate of birds.
Smooth muscles attached to feather follicles help erect the feathers and expose
the skin surface to permit still higher rates of evaporative cooling to combat the
increased heat production of flight. With increased mobility, Aves have been
able to inhabit a wide variety of environments, necessitating integumentary
adaptive plasticity, while being restricted to their basic phylogenetic pattern so

crucial for flight. In Section 3.3.1 we examine how this is achieved by
modulating the permeability barrier to permit survival under xeric stress, to
compensate for the unpredictable availability of water, and to meet
thermoregulatory requirements.
3.3.1
The Sebokeratinocytes and the Avian Permeability Barrier
Except for an inherently high lipogenic activity of the epidermis [29], the basic
avian scheme of epidermal proliferation—progressive increase in differentiation
markers such as keratin and terminal differentiation into non-viable corneocytes
—is similar to that of mammals. Free, non-membrane-bound lipid droplets
appear from the basal layer onward; and as the cells stratify and differentiate,
there is an increase in both the size and numbers of the lipid droplets, the
quantitative measure of which varies in different body areas. Epidermis from
specific sites (such as the toe web, the wattles and combs, or the maxillary rictus,
as well as areas devoid of feathers) shows a higher degree of lipogenesis [30].
However, in the upper stratum transitivum (equivalent to mammalian SG, but
lacking keratohyalin granules), large multilamellar or multigranular bodies of
varying morphological appearance (Fig. 5, inset (MGBs)) are also synthesized.
In the normal course, these MGBs undergo dissolution and transformation in
situ into lipid droplets, fuse with the other lipid droplets, and in differentiated
corneocytes, form one or several large lipid pools at the core of the cell (Fig. 5).
The lipids eventually escape from the corneocyte (through breaks in membranes
or porosities) into the extracellular domains of SC. Alternatively, some MGBs
are retained as lamellar lipids within the core of the corneocyte cytosol. The fate
of MGBs depends on the environment, as has been shown in work with early
nestling life [31], water deprivation stress in adult zebra finches [32], and heat-
and cold-acclimated pigeons [33]. Under these conditions, when conserving body
water takes precedence over evaporative cooling, the MGBs are secreted at the
stratum transitivum-SC interface. Their disk contents fuse together as in
mammals, forming occlusive lipid bilayers that provide an effective permeability
barrier, substantially decrease the TEWL, and aid in water conservation. This
facultative waterproofing is a dynamic response of the skin, since providing
drinking water results in the breakdown and transformation of nascent MGBs
into lipid droplets within a day. This is vie wed as a prelude to restoring the basal
barrier organization that allows for increased TEWL and evaporative cooling [34].
Further specializations in the skin providing additional thermoregulatory
capabilities are seen in many species of vultures and storks, where a reduction or
loss of feather s on the head and/or neck creates an exposed patch of skin that is
well vascularized and often retractable (e.g., neck of vultures). More often, such
bare patches acquire bright coloration (for intra-orinterspecific visual
communication), owing to red carotenoids accumulated in the basal epidermal
186 MENON
FIGURE 5 Avian epidermis: ultrastructural features of stratum transitivum (ST) and SC.
Uppermost cell in ST of the zebra finch shows multigranular bodies (MGB), electron-
lucent lipid inclusions (L), and multilamellar bodies breaking down to form electron-
lucent lipid droplets in situ, before cornification. (Modified from Ref. 40.) (Upper inset)
Corneocytes showing overlapping, tapering ends of individual cells and large core of
retained lipids in the middle of individual corneocytes (L). (Lower inset) Another
structural variation of the MGBs (OsO4 postfixation).
layer, and these areas provide some protection against UV exposure. Skin lipids
also may be the basis of the toxins in birds such as the pitohui, where they serve
a chemical defense function against predators [35].
3.4
Adaptations for Land-to-Water Transition
Many species of vertebrates have retraced the phylogenetic pathways, returning
to residence in an aquatic environment, while retaining aerial breathing. These
include reptiles (marine iguanas, sea snakes, turtles, crocodiles) water birds, and
mammals (seals, otters, hippos, cetaceans). Most return to land for breeding, the
exception, cetaceans, are truly aquatic. Among the marine mammals, the skin of
pinnipeds, sea otters, and so on is largely similar to that of terrestrial mammals;
but cetacean skin (whales and porpoises) shows many remarkable adaptations to
marine life.
The challenges of aquatic life include maintaining the barrier to efflux and
influx of water in a fully hydrated integument, whereas in terrestrial forms,
hydration disrupts the stratum corneum cohesiveness and organization of barrier
lipid structures. Snake skin has its barrier localized in the mesos layer—
sandwiched between the outer and inner layers, and possibly protected from
water damage by a nonwettable surface—owing to microornamentation and
surface sculpturing that could trap a layer of air and waterproof the surface. Even
so, sea snakes shed their skin with much higher frequency than their terrestrial
counterparts. A secretion of lipid film to the skin surface in sea snakes has been
alluded to, but not convincingly demonstrated.
In birds, the feathers are inherently nonwettable and further are aided by
secretions of the preen gland, applied to feathers by active preening. The exposed
parts such as the web and scales on feet have a high lipid secretion—thus aquatic
birds have a certain protoadaptation for this mode of life.
For truly aquatic mammals like cetaceans, additional challenges in
integumentary adaptations are achieving hydrodynamic efficiency at the
boundary layer, maintaining body temperature, and barrier, and prevent
biofouling by attachment of sessile organisms and their larvae. The dermis and
blubber are indeed considered to be special adaptations, but it is the epidermis
that displays the most obvious structural and biochemical adaptations in terms of
barrier functions in cetaceans that evolved over 55 million years. It is instructive
to see how they evolved by first examining an amphibious mammal, the hippo,
which is genetically the closest relative of the whales.
3.4.1
The Skin of the Hippopotamus: Amphibian Adaptations in a
Mammal
Hippos spend most of their time in water. They enter their habitat (mud, stagnant
water, or clear waters of lakes and rivers) before sunrise and remain there with
188 MENON
their head and back above, till the full heat of the day. They emerge to bask in
the sun for about an hour, before returning to the water, and only after sunset do
they get out to graze on the land. Since hippos rely on the land for food, they are
truly amphibious. Most adults show heavily scarred skins from fights with others,
but the dermis, as mentioned earlier, is specialized for a high degree of impact
resistance [36]. The skin comprises about 18% of the animal’s body weight
(270kg for an adult weighing about 1500kg), has a thickness of about 300cm,
and is smooth and almost hairless (20–30 short, fine hairs per 100cm2). A unique
feature is the presence of subdermal glands, whose ducts open on the skin
surface, 2 to 3cm apart. The epidermal-dermal junction shows deep rete pegs.
The epidermis on the back is about 0.7 to 1.0 mm thick. The nucleated layers
show the presence of keratin filaments, desmosomes, and some melanin granules.
Occasionally, small lipid droplets are also seen in the cytoplasm. The SG is ill
defined because keratohyalin grannles (KHGs) are atypical and small, and not
densely packed as in terrestrial mammals. Both the SG and SC are reportedly
Positive to periodic acid-Schiff (PAS) staining [36], indicating the presence of
mucoid substances or retention of glycolipids. At the SG-SC junction, secreted
lamellar body contents are seen, although these are much less conspicuous than
in terrestrial mammals (Menon and Drewes, unpublished). Corneocytes show
regular interdigitations and many desmosomes, and their cytosol is filled with
keratin filaments that impart a uniform electron density (Fig. 6). Hence the SC is
more keratinized than in the truly aquatic cetaceans (see Sec. 3.4.2).
From an adaptive perspective, the skin of the hippo is unique in its paucity of
sebaceous lipids and in the copious secretions from the subdermal “sweat”
glands that cover the skin surface. When in the sun, the “sweat” dries and
provides a dry film, possibly retarding water loss to some extent, as in
amphibians. The secretion, owing to its putative porphyrin content, is known to
undergo a color change when exposed to sunlights, and it is speculated to have
some UV protection value. Besides, the secretion may have potential
antimicrobial and antibiofouling functions. The retention of glycoconjugates in
the epidermis and SC would make the tissue more hydrophilic, hence compatible
with the fully hydrated state of the hippo’s amphibious mode of life.
3.4.2
Cetacean Skin: Truly Aquatic Adaptations
The cetacean skin is characterized by a general lack of hair follicles, and
sebaceous and sweat glands; it has a very thick (1.0–3.5mm) epidermis.
However, hair is present in fetal life, and a few hairs are retained on the snout in
some species, as are remnants of hair follicles and sebaceous structures [37]. The
dermis is thick, and rete ridges or epidemal-dermal interdigitations are very
deep, (Fig. 7a left). The hypodermis is exceptionally thick, owing to the presence
of blubber.
Despite some differences between the skin of toothed whales and baleen
whales, the epidermis is similar in both suborders, although thicker in the large
whales than in small toothed whales [38]. In the typical cetacean, epidermis is
divisible into the stratum basale, stratum spinosum, and a parakeratotic stratum
externum (equivalent to stratum corneum). There is no recognizable stratum
granulosum. Because of the large numbers of deep rete pegs, cetacoans have
many stratum basale cell relative to terrestrial mammals. Basal cells have
interdigitating cell membranes, desmosomes, a high nucleocytoplasmic ratio, an
abundance of ribosomes, mitochondria, and perinuclear lipid droplets, but
relatively few keratin filaments. Lipid droplets and keratin filaments increase
progressively in number through the spinous layer (Fig. 7 right). The keratin
content of the cells is always in the form of dispersed, nonaggregated filments,
and there is a conspicuous absence of keratohyalin granules. Interspersed with
the keratin filaments are innumerable lamellar bodies and large lipid droplets of
varying electron density. Histo- chemical observations suggest that the
membrane domains of basal cells contain predominantly acidic lipids, while
neutral lipids predominate in the suprabasal cells—mainly cytosolic lipid
droplets. When stained for glycoconjugates, the cytoplasm (but not membranes)
of the spinous layer shows positive staining. However, in the stratum externum,
the membrane domains show such staining for glycoconjugates, coupled with a
loss of cytosolic staining, correlating with the secretion of lamellar bodies that is
seen at the electron microscopic level (Fig. 8).
Extensive rete pegs ensure the integrity of epidermis subjected to constant
physical shearing forces exerted by the water flow at the boundary layer. It also
provides for a large number of cells in the germinative zone (stratum basale) that
generate an extremely thick epidermis with substantial, continuous loss through
exfoliation. The keratins of cetacean epidermal layers remain to be biochemically
characterized and compared with those of terrestrial mammals. The abundance
of lamellar bodies (LBs), however, rivals that seen in terrestrial mammals, but
the LB secretion is not as obvious in cetaceans because of the gradual transition
of the spinous layer into the parakeratotic stratum externum without an
intervening stratum granulosum. Since keratohyalin granules are not present, the
keratinization pattern is also different—the keratin filaments remain
nonaggregated and sparsely distributed within the cytosol of the terminally
differentiated cells. These cells also contain pycnotic nuclei, lipid droplets of
considerable dimensions, and melanin granules. The cornified envelopes are
rather thin, and no retained lamallar bodies are seen in the cytosol. This is
indicative of active lamellar body secretion prior to terminal differentiation.
Since, however, only sparse contents of LBs are seen in the extracellular
domains of stratum externum, a continuous and ongoing extrusion and loss of
these lipids into the boundary layer water may be surmised. The glycoconjugate
staining in the membrane domains of stratum externum also suggests that the LB
contents are not fully processed—that is, the sugars are not cleaved off the
190 MENON
FIGURE 6 Amphibious mammal: portions of SG and SC of hippo epidermis. An

occasional lipid droplet (Ld) is seen in corneocytes. Keratohyalin (KH) granules are not
as dense as in terrestrial forms (OsO4 postfixation). (From Menon and Drewes,
unpublished).
glycosphingolipids to generate the ceramide-rich, nonpolar bilyers as in
terrestrial mammals.
In evolving adaptations to the marine habitat, cetacean epidermis acquired
remarkably unique features in its structural and biochemical makeup. The
FIGURE 7 Marine mammal. (Left) Light microscopic view of the skin of the harbor
porpoise, showing rete pegs (arrows); lipid droplets in the highly stratified epidermis
stained with Fat Red 7B, a neutral lipid-specific stain. (Modified from Menon et al. J Cell
Tissue Res 1986; 244:385–394.) (Right) Higher magnification of the epidermal cells from
the same specimen showing the coalescence of smaller lipid droplets as the cells stratify
and move upward (0.5µm thick plastic sections). (Modified from Ref. 40 with permission
from John Wiley & Sons, Inc.)
permeability barrier function, though not quantifiable by conventional
techniques, must depend on the lamellar body secretory system. Adaptive
modifications in this regard may lie in the lack of postsecretory processing, so
that the more polar glycolipids are retained throughout the stratum externum,
making this layer compatible with a highly hydrated state, as is the oral
192 MENON
FIGURE 8 Higher magnification view of two adjacent corneocytes of cetacean epidermis

showing sparsely distributed keratin filaments in cytosol and sparse intercellular lipid
lamellae (ICL). (Modified from Ref. 40 with permission from John Wiley & Sons, Inc.)
(Upper inset) Light microscopic appearance of the cetacean stratum externum (SE), with
individual corneocytes displaying lipid droplets (LD) of varying size. (Lower inset) High
magnification of portion of cytosolic lipid droplet (LD), as well as secreted lamellar body
contents within the extracellular domains of corneocytes (curved arrow). (Modified from
Menon et al., 1986).
epithelium of terrestrial mammals [39]. It has been proposed that the glycolipids
secreted into the boundary layer may reduce turbulence, enhancing
hydrodynamic efficiency [39, 40]. Another function of the secreted lipids and
enzymes from the LBs is to provide a barrier against biofouling. The “zymogel,”
or an extracellular aggregate-attached enzyme, hydrolyzes the adhesive proteins
of biofouling organisms that tend to settle on the skin of whales [41].
The role of cytosolic lipid droplets within the stratum externum also deserves
to be examined in detail. Elias et al. [40] proposed that their functions include
providing increases in buoyancy and cellular energetics, or generation of
glycerol, an antifreeze compound. The presence of these droplets within a cell
with effete keratin filaments may also help in small deformations of the surface
of individual cells (acting like bean-bags) to conform to the boundary layer flow
for increased hydrodynamic efficiency. It is also possible that the lipids ooze out
of porosities in the cell envelope (similar to what is seen in avian
sebokeratinocytes), thereby coating the cell surface. The surface sculpturing of
individual corneocytes seen with conventional scanning electron microscopes
[39] was not prominent when Baum et al. [42] used freeze preservation for
retention of the surface coating, and hence a much smoother surface—possibly

correlated to decreased surface wetting.
To sum up, lipogenesis and lipid secretion, used uniformly for permeability
barrier on land, have been successfully adopted for a fully aquatic life, by
altering the types of lipid synthesized, and postsecretory modification of barrier
lipids, and thereby the wettability of corneocytes. The modified pathway of
keratinization (i.e., by deleting the keratohyalin granules or profilaggrin),
possibly reflects the redundancy of natural moisturizing factors (derived from
proteolysis of filaggrin in terrestrial mammals) in cetaceans.
4
CONCLUSIONS
Comparative biology illuminates the phenomenal adaptive plasticity of the
vertebrate integument. The facultative waterproofing ability of tree frogs,
reptiles, and birds discussed in the preceding sections is only one of the many
attributes of this plasticity. Many other features not addressed here for reasons of
space include production and secretion of antimicrobial peptides by
keratinocytes, skin glands in lower vertebrates and sweat glands in mammals,
pheromones and other molecules used for intra- and interspecies
communications, and the use of cryptic and warning colorations. Human skin,
the most pampered (cosmetics, cleansers, topical medications, controlled
microclimate) and most abused (environmental pollution, toxic exposure in many
forms of activities such as agriculture and industrial production), is also the most
highly evolved integumental tissue.
We are the only species exploring the environments of land, air, deep sea, the
arctic, and space. To what extent our species exhibits facultative changes in the
barrier functions is presently not clear, although site-specific and interindividual
variations in TEWL may be indicato of such fine-tuning of the epidermal barrier.
Humans have also “created” artificial skin through tissue engineering for clinical
applications and for research (Fig. 9), which, as a biotech industry, is a growing
commercial enterprise. With the advent of gene therapy, we would except to see
the use of autologous, genetically modified skin grafts as a “bioreactor” [43] to
secrete growth factors, hormones, and drugs into the circulation. As creators, we
could learn much from evolution and evolutionary adaptation in other life-forms
—saving us from having to reinvent the wheel. Those working in the challenging
field of transdermal drug delivery would greatly benefit from a “biomimetic”
approach and an understanding of how the integumentary system responds to
different degrees of barrier disruption [44].
ACKNOWLEDGMENTS
I express my gratitude to fellow skin biologists who have taught, encouraged,
and inspired me to work in this field during the past two decades. Among them,
194 MENON
FIGURE 9 Ultrastructure of “created skin”: a commercially available skin equivalent

shows vacuolization in viable cells, paucity of keratohyalin, very few keratin filaments in
SG, and less than perfect differentiation of the SC.
Drs. A.M.Lucas, W.B.Quay, L.F.Baptista, and B.Forslind are no longer with us.
Drs. P.M.Elias, P.F.A.Maderson, P.Stettenheim, R.C.Drewes, and S.Grayson are
thanked for many years of collaborations, long hours of stimulating and animated
discussions over gourmet meals or nature walks, friendship, and tolerance for my
many unfocused ideas. Collaborations with R.B.Lillywhite and C.Pfeiffer and
discussions with N.Kitson and S.B.Hoath have been very rewarding. At the
University of Baroda in India, Dr. R.V.Shah initiated me in research, and a Homi
Bhabha Fellowship as well as support from the visionary Vice-Chancellor
Dr.B.C.Parekh, (presently member of the House of Lords, U.K.) shaped my
research career. Dr. Jaishri Menon supported me through my many “sabbaticals”
from home, and also provided microscopy facilities when in dire need.
J.Bouwstra, G.Behrmann, L.Halkier-Sorensen, J.Dumbacher, S.H.Lee,
W.Holleran, and L.Norlén have freely shared with me unpublished manuscripts
and new information. Sheri Lenc provided invaluable editorial help.
REFERENCES
1. Chuong CM, Nickloff BJ, Elias PM, Goldsmith LA, Macher E, Maderson PFA,
Sundberg JP,Tagami H, Plonka PM, Thestrup-Pederson K, Bernard BA, Schroder
JM, Dotto P, Chang MHC, Williams ML, Feingold KR, King LE, Kligman AM,
Rees JL, Christophers E. What is the true function of skin?. Exp Dermatol 2002;
11:159–187.
2. Jablonski NG, Chaplin G. The evolution of human skin coloration. J Hum Evol
2000; 39:57–106.
3. Jarman PJ. On being thick-skinned: dermal shields in large mammalian herbivores.
Biol J Linalan Soc 1989; 36:169–191.
4. Shadwick RE, Russell AP, Lauff RF. The structure and mechanical design of
rhinoceros dermal armor. Phil Trans R Soc Lond B 1992; 337:419–428.
5. Menon GK, Elias PM. The epidermal barrier and strategies for surmounting it. In:
Hengge UR Volc-Platzer B, eds. The Skin and Gene Therapy, Springer Verlag,
Berlin 2001; 337:3–26.
6. Menon GK, Brandsma J, Schwartz P.Gene gun and the human skin: ultrastructural
study of the distribution of gold particles in the epidermis. J Invest Dermatol 1997;
110:637 (abstr).
7. Lévêque JL. Measurement of transepidermal water loss. In: Lévêque JL. ed.
Cutaneous Investigation in Health and Disease. Noninvasive Methods and
Instrumentation, New York: Marcel Dekker, 1989; 337:135–152.
8. Hadley NF. Lipid water barriers in biological systems. Prog Lipid Res 1989; 28:1–
33.
9. Menon GK, Ghadially R. Morphology of lipid alterations in the epidermis: a
review. Micros Res Techniques 1997; 37:180–192.
10. Elias PM, Cullander C, Mauro T, Rassner U, Komuves L, Brown B, Menon GK.
The secretory granular cell: the outermost granular cell as a specialized secretory
cell. J Invest Dermatol Symp Proc 1998; 3:87–100.
11. Holbrook K. Ultrastructure of the epidermis In: In: Leigh IM Lane BE Watt FM
The Keratinocyte Handbook, Oxford: Cambridge University Press, 1994; 3:3–39.
12. Elias PM, Friend D. Permeability barrier of mammalian epidermis. J Cell Biol
1975; 65:180–191.
196 MENON
13. Packard MJ, Packard GC, Willard R, Tucker JK. The role of the integument as a
barrier to penetration of ice into overwintering hatchlings of the painted turtle
(Chrysemys picta). J Morphol 2000; 246:150–159.
14. Warner RR, Boissy YL, Lilly NA, McKillop K, Marshall JA, Stone KJ.. Water
disrupts stratum corneum lipid lamellae: damage is similar to surfactants. J Invest
Dermatol 1999; 113:960–966.
15. Lillywhite HB, Mittal AK. Amphibian skin and the aquatic-terrestrial transition:
constraints and compromise related to water exchange. In: In: Mittal AK, Eddy FB,
Datta Munshi JS, Water/Air Transition in Biology, New Delhi: Oxford & IBH
Publishing, 1999; 113:131–144.
16. McClanahan LL, Stinner JN, Shoemaker VH. Skin lipids, water loss and energy
metabolism in a South American tree frog (Phyllomedusa sauvagei). Physiol Zool
1978; 51:179–187.
17. Drewes RC, Hillman SS, Putnam RW, Sokol OM. Water, nitrogen and ion balance
in the African tree frog Chiromantis pertersi Boulenger (Anura, Racophoridae)
with comments on the structure of the integument. J Comp Physiol 1977; 116:257–
267.
18. Kattan GH, Lillywhite HB. Humidity acclimation and skin permeability in the
lizard Anolis carolinensis. Physiol Zool 1989; 62:593–606.
19. Maderson PFA, Rabinowitz T, Tandler B, Alibardi L.. Ultrastructural contributions
to an understanding of the cellular mechanisms involved in lizard skin shedding
with comments on the function and evolution of a unique lepidosaurian
phenomenon. J Morphol 1998; 239:1–24.
20. Maderson PFA. The squamate epidermis: new light has been shed. Symp Zool Soc
Lond 1984; 52:111–126.
21. Maderson PFA, Zucker AH, Roth SI. Epidermal regeneration and percutaneous
water loss following cellophane stripping of reptile epidermis. J Exp Zool 1978;
204:11–32.s.
22. Landmann L. Bereiter-Hahn J. Matoltsy AG. Richards KS. The skin of reptiles:
epidermis and dermis. Biology of the Integument, Vol 2: Vertebrates, Springer-
Verlag, Berlin, 1986; 204:150–187.
23. Tu MC, Lillywhite HB, Menon JG, Menon GK. Postnatal ecdysis establishes the
permeability barrier in snake skin: new insights into barrier lipid structures. J Exp
Biol 2002; 205:3019–3030.
24. Tucker R. The digging behavior and skin differentiation in Heterocephalus glaber.
J Morphol 1981; 168: 51-71.
25. Daly TJM, Buffenstein R. Skin morphology and its role in thermoregulation in
mole-rats, Heterocephalus glaber and Cryptomys hottentotus. J Anat 1988; 193:
495-502.
26. Hildebrand M. Analysis of Vertebrate Structure. 3d eds. New York: John Wiley &
Sons, 1988.
27. Lucas AM, Stettenheim PR. Avian Anatomy: Integument. Agriculture Handbook
362, US Department of Agriculture, Washington, DC, 1972.
28. Stettenheim PR. The integumentary morphology of modern birds: an overview. Am
Zool 2000; 40: 461-477.
29. Menon GK. Glandular functions of avian integument: an overview. J Yamashina
Inst Ornithol 1984; 15: 166-177.
30. Menon GK, Menon JG. Avian epidermal lipids: functional considerations and
relationship to feathering. Am Zool 2000; 40: 540-552.
31. Menon GK, Baptista LF, Elias PM, Bouvier M. Fine structural basis of cutaneous
water barrier in nestling zebra finches Poephila guttata. Ibis 1988; 130: 503-511.
32. Menon GK, Baptista LF, Brown BE, Elias PM.. Avian epidermal differentiation:
II. Adaptive response of permeability barrier to water deprivation and
replenishment. Tissue Cell 1989; 21: 83-92.
33. Peltonen L, Arieli Y, Pyornila A, Marder J. Adaptive changes in epidermal
structure of the heat-acclimated rock pigeon (Columba livia): a comparative
electron microscopy study. J Morphol 1998; 235: 17-29.
34. Menon GK, Maderson PFA, Drewes RC, Baptista LF, Price LF, Elias PM.
Ultrastructural organization of avian stratum corneum lipids as the basis for
facultative cutaneous waterproofing. J Morphol 1996; 227: 1-13.
35. Dumbacher JP, Pruett-Jones S. Avian Chemical Defense. In : NolanV, Ketterson
ED eds. Current Ornithology, NewYork:Plenum Press, 1996: 137-174.
36. Luck CP, Wright PG. Aspects of the anatomy and physiology of the skin of the
hippopotamus (H. amphibius.). Q J Exp Physiol Co Med Sci 1964; 227: 1-14.
37. Behrmann G. Natural skin protection of whales (Cetacea) Lebensraum. "Meer
Nordseemuseum Bremenhaven H.23, 2001.
38. Pfeiffer CJ, Jones FM. Epidermal lipid in several cetacean species: ultrastructural
observations. Anat Embryol 1993; 188:209-218.
39. Pfeiffer CJ, Menon GK. Cellular ultrastructural and biochemical specializations in
the cetacean epidermis. In: Pfeiffer, CJ Molecular and Cell Biology of Marine
Mammals, Malabar, FL, Krieger, 2002: 396-411.
40. Elias PM, Menon GK, Grayson S, Brown BE, Rehfeldt SJ. Avian sebokeratocytes
and marine mammal lipokeratinocytes: structural, lipid biochemical and functional
considerations. Am J Anat 1987; 180: 161-177.
41. Baum C, Meyer W, Roessner D, Siebers D, Fleischer LG. A zymogel enhances the
self-cleaning abilities of the skin of the pilot whale (Globicephala melas). Comp
Biochem Physiol 2001; 130(4): 835-847.
42. Baum C, Stelzer R, Meyer W, Siebers D, Fleischer LG. A cryo-scanning electron
microscopic study of the skin surface of the pilot whale (Globicephala melas.).
Aquat Mammals 2000; 26(1):7–16.
43. Cao T, Tsai SY, O’Malley BW, Wang XJ, Roop DR. The epidermis as a bioreactor:
topically regulated cutaneous delivery into the circulation. Hum Gene Ther 2002;
13:1075–1080.
44. Menon GK. New insights into skin structure: scratching the surface. Adv Drug
Deliv Rev 54(suppl 1):2002; S3–S17.
9
Skin Barrier Function in Diseased Skin and in
Normal Skin Exposed to Delipidizing
Compounds: A Skin Penetration Perspective
Anders Boman and Magnus Lindberg
Stockholm, Sweden
The skin is constantly exposed to environmental factors that might influence the
integrity of the skin barrier. Chemicals and pharmaceuticals can penetrate into
the skin and have a local effect, or they may penetrate the skin, enter the
circulation, and exert a general effect. In penetrating the skin they may also be
altered by local metabolism. In toxicology, the percutaneous route for entrance
of chemicals into the body has been underestimated [1]. For example, penetration
through the skin is the main route by which pesticides enter the body [2]. Today
it is recognized that stratum corneum is the main barrier to penetration and that
the main route for penetration into and through the skin is via the lipid-rich
intercellular space of stratum corneum [3]. Skin diseases characterized by
disturbed keratinization, (e.g., psoriasis, ichthyosis) or by changes in the lipid
composition (e.g., atopic dermatitis) do have a detectable defect in the barrier
function. A common environmental exposure (at work, at home, and in the
community) is to different types of solvent. This chapter discusses both
skin diseases in which barrier properties are altered and the effects of solvents.
The development of systems for percutaneous drug delivery, which calls for the
addition of penetration enhancers to a product, [3–6], is not extensively explored
in this text.
1
PERCUTANEOUS ABSORPTION
Absorption, or penetration through the skin, is a passive process. It is usually
described by Fick's first law of diffusion:
where Jss is the permeation rate at steady state, Kp the permeability coefficient,
and C the concentration gradient over the membrane.
This is a simplified model which has been shown to be applicable in many
cases [1, 2], However, it does not take into account local effects in the skin or the
possibility of local metabolism or accumulation (stratum corneum acting as a
200 BOMAN AND LINDBERG
reservoir). The intercellular lipids in stratum corneum are described as being be

organized into various levels of functional structure with alternating lipophilic
and hydrophilic domains [7–20]. Exposing the skin to lipophilic and amphiphilic
compounds not only gives them an opportunity to diffuse into the skin and
further into the circulation but also may permit them to influence the barrier
properties of the skin. Exposure to lipophilic and amphiphilic substances such as
organic solvents, oils, or surfactants leads to insertion of small molecules into
these lipophilic domains, subsequently altering the structured organization of the
intercellular space. The results are increased disorganization and altered barrier
function. Since the intercellular lipid matrix in the stratum corneum is composed
of both free and chemically bound lipids, parts of it can be extracted by
lipophilic substances or surface active agents. This may in turn lead to increased
diffusion of compounds (e.g., water) through the skin [5, 6]. Exposing the skin to
chemicals of that type may also alter the water-holding capacity of stratum
corneum. A loss of or a change in barrier function may also lead to increased
bioavailability and absorption [21].
Several factors influence percutaneous absorption and may play various roles
in different dermal exposure situations. Upon dermal exposure, the
physicochemical properties of the compounds the skin is exposed to may be of
more importance and may contribute more to the local effects than such
environmental factors as vehicle, temperature, and biological properties.
Important physicochemical properties in this case one molecular volume and
molecular weight, lipophilicity, and volatility (i.e., boiling point and vapor
pressure).
The barrier properties of the skin are determined by the delicately balanced
mosaic of corneocytes and lipids which under normal situations provides a
perfect barrier to sustain human life in dry air [22]. However, in several skin
diseases this balance is disturbed, and a perturbed barrier function and a
subsequent increase in transepidermal water loss (TEWL) can be detected.
Atopic dermatitis, psoriasis, and ichthyosis have been especially well studied in
this respect. Involuntary exposure to industrial and household chemicals may
also influence the lipids in the skin barrier, hence transepidermal water loss and
penetration into the skin of other environmental compounds.
2
SKIN DISEASES
It has been demonstrated that the skin as a barrier is defective in some diseases.
However, the implication of this, in adequacy, especially with regard to the
possible penetration and altered effects of locally applied dermatological
pharmaceuticals and environmental factors, has been addressed only to a minor
extent (e.g., Refs. [23, 24]). Another aspect of skin diseases is treatment with
moisturizers, which have potential to interact with the structures of stratum
corneum, hence also potential to affect skin function.
TABLE 1 Properties of Some Common Solvents
A SKIN PENETRATION PERSPECTIVE
201
a , Completely miscible with water; i.s., practically insoluble in water.

Source: Data from Refs 2–8.
A SKIN PENETRATION PERSPECTIVE 203
2.1
Atopic Skin
Measuring the transepidermal water loss across skin in persons with atopic
dermatitis will reveal that there is an increase in the diffusion of water across the
barrier. There is evidence that this increase is primarily due to a difference in
composition and organization in the intercellular lipids, mainly differences in
cholesterol and ceramide content [25–36].
The barrier property of the skin in atopic persons is easily disturbed by exposure
to lipid-extracting and hydrophobic compounds, leading to an increased
susceptibility to irritant compounds [37–42]. This is demonstrated, clinically by
noting that the risk of developing hand eczema in certain occupations is higher
among persons with atopic dermatitis than in the normal population [43, 44]. An
abnormal stratum corneum is also noticed in experimental work: it has been
shown that atopic skin has a lower water-holding capacity than normal skin
[45,46], as manifested clinically xerosis in [47].
2.2
Psoriatic Skin
Ultrastructural studies reveal a different organization of the intercellular lipids in
psoriatic placques [48–51]. However, water diffusion through and retention in
the plaques is not increased over what is seen in normal control sites [45],
implications of this can be further evaluated.
2.3
Ichthyosis
The Ichthyoses are a group of keratinization disorders of differing etiological
backgrounds [52]. A common characteristic is that the structure and organization
of the intercellular space of stratum corneum is abnormal and is associated with
an increased TEWL. In some instances this defective skin barrier seems to be
associated with alterations in cholesterol metabolism in the skin and in the
organization of the lamellar structure of the intercellular lipids [53–57]. The
abertion in cholesterol sulfate found in the skin in of persons with X-linked
ichthyosis is also present in their hair and nails [58]. Increased susceptibility to
irritant trauma has also been reported [59].
3
TREATMENT WITH MOISTURIZERS
Moisturizers are extensively used in the treatment of skin diseases, especially
eczema, psoriasis, and ichthyosis. They are also used on dry skin and as
cosmetics and skin care products [60]. Moisturizers used today are mainly
composed of lipids in different mixtures with an addition of such natural
moisturing factors (NMFs), as urea, lactic acid, and sodium chloride. It is
thought that lipids and/or NMFs penetrates the intercellular lipid phase of stratum
corneum, and it has been shown that the local application of moisturizers can
alter the barrier properties in normal skin, both in irritant contact reactions
[61–66] and in atopic dermatitis [61, 67]. The lipid composition of a moisturizer
is one factor that determines its effect on the barrier [68, 69], and these
compounds, can both increase and decrease penetration of other substances [70, 71]
.
4
DELIPIDIZING AGENTS (SOLVENTS)
Several experimental studies have addressed the problem of irritation from organic
solvents. The mechanism of irritation by solvents is dependent on a variety of
factors. One is the interaction of a solvent with the structured lipids in the stratum
corneum and the lipid film on the skin surface. In Table 1 and Figure 1 it can be
seen that solvents vary chemically and that the majority of the commonly used
solvents are highly lipophilic. Several have a high octanol/water partition
coefficient, and this makes them extremely potent delipidizing agents. Exposure
to the skin invariably influences the surface and intercellular lipids in the stratum
corneum.
The first visible reaction to a single topical exposure to some solvents is
whitening of the skin. Human skin exposed to a series of different solvents
showed immediate whitening except for dimethyl sulfoxide and 1,2-propanediol.
This result can be attributed to conformational changes and extraction of the
surface and intercellular lipids upon contact with the solvents [72, 73].
Repeated contact with solvents extracts more surface and intercellular lipids,
leaving the skin with reduced water protection and water-retaining properties. This
leads to increased water diffusion and the loss of water, followed by dehydration
of the skin, which will be felt as dryness and chapping. The reduced-water
holding capacity and increased water diffusion can be measured as a decrease in
skin impedance and increase in transepidermal water loss [74–80]. The water
barrier can be regenerated after solvent extraction within 15 to 20 days.
Delipidizing properties of solvents vary. Most effective is a 2:1 mixture of
chloroform and methanol. This mixture is often used in experimental lipid
extraction and delipidizing of the skin [81–83]. The efficacy in lipid extraction
of solvents is graded in the following order: chloroform:-methanol (2:1) >
chloroform > diethyl ether > acetone > ethanol [84– 86]. However, Abrams and
coworkers found no correlation between lipid extraction and increase in water
loss [83]. After exposure of human skin in vitro to a series of solvents or solvent
mixtures, these investigators followed transepidermal water loss and also
measured lipid extraction. Chloroform-methanol and hexane-methanol mixtures
FIGURE 1 Names and structural formulas of some commonly used organic solvents.
extracted comparable amounts of lipids of various classes, but only chloroform-

methanol induced a significant increase in transepidermal water loss. Length of
exposure was also found to be of significant importance. A longer exposure time
extracted more lipids and gave a larger increase in transepidermal water loss.
Repeated exposure to solvents will generally lead to irritant dermatitis with
erythema and edema formation. However, exposure to organic solvents will in
some cases lead to immediate flare-up of erythema, commonly characterized by
its intensity [23, 87–90]. The development of laser Doppler flowmetry has
facilitated the study of erythema formation and represents a noninvasive,
objective test method. Several solvents has been investigated with this technique
[72, 91, 92]. The technique verified the variation in erythema-forming properties
of the solvents that had been assessed with subjective methods [89].
A relationship between exposure time and erythema intensity was seen in one
study for trichloroethylene and dimethyl sulfoxide. There was also a clear
difference in kinetics in the development of erythema following exposure to
these solvents. Onset, intensity, and duration varied considerably. Dimethyl
sulfoxide and trichloroethylene gave the largest increase in blood flow values
and the longest duration. Several solvents did not induce any increase in blood
flow value [72]. Persistent effect (past 60 h) was found for toluene, n-hexane,
and n-butanol [92].
Wahlberg used a skin fold measuring technique to study the edemaforming
properties of organic solvents after repeated contact in guinea pigs and rabbits
[93]. Daily exposures to solvents were combined with measurements of skin fold
thickness, and marked differences in edema-forming properties were noted in the
seven solvents studied. Trichloroethylene and toluene had the most conspicuous
effect, resulting in doubled skin thickness after 10 days of exposure. The least
effect was seen for ethanol and methyl ethyl ketone, with only a minor increase
during the exposure period. This coincides well with the graded order for
irritating properties of solvents [94].
5
CLINICAL EXPERIENCE: CONTACT DERMATITIS
It is known from clinical and experimental experience in man that organic solvents
are potent irritating chemicals, capable of inducing irritant contact dermatitis
upon contact with unprotected skin [23, 72, 83, 95–101]. The irritating capacity
of solvents follows this order: aromatic > aliphatic > chlorinated > turpentine >
alcohols > esters > ketones. This result can be attributed partly to volatility and
mainly to lipophilicity [94].
Skin exposure to solvents will result in erythema, scaling, and dryness,
eventually evolving into eczema. Usually the hands are involved, but owing to
the volatility of the solvents the face and neck can also be affected [102].
Outbreaks of irritant dermatitis from various causes can occur. Nethercott and
coworkers showed that wearing coveralls damp with solvents after dry cleaning
resulted in epidemic outbreak of irritant dermatitis in a factory [103].
Several factors influence the irritant action of solvents. Boiling range and
irritation effect were correlated in a series of petroleum solvents. Fractions in the
lower boiling range were more irritating than those from higher boiling ranges,
and solvents with aromatic components more irritating than those with aliphatic
[96]. This finding may be related to differences in lipid extraction properties and/
or penetration rates.
Concentration of the solvent applied is also an important factor in the irritant
action of solvents. The eczema-developing propensity of solvents has also been
shown in epidemiological studies [104–106] Solvents are seen as one of several

important factors in inducing irritant contact dermatitis in many occupations.
6
CONCLUSIONS
A normal structure and function of the barrier, stratum corneum, is vital for the
organism. Environmental factors, pharmaceuticals, and chemicals may interfere
with both the structure and the homeostasis of the barrier and consequently alter
its function, hence the possibility of percutaneous penetration. Inflammatory skin
disorders and skin conditions with changes in keratinization also lead to altered
barrier function and penetration characteristics. Although our knowledge in this
area has increased during the past decade, there is still a need for more
information on the structure of stratum corneum and how it is controlled and
regulated in healthy and diseased skin. This is a requirement for an improved risk
assessment but also is necessary for the development of new pharmacological
treatments and treatment systems.
REFERENCES
1. Roberts MS, Walters KA. Dermal Absorption and Toxicity Assessment, New York:
Marcel Dekker, 1998.
2. Nielsen JB, Nielsen F. Dermal in vitro penetration of methiocarb, paclobutrazol,
and pirimicarb. Occup Environ Med 2000; 57:734–737.
3. Schaefer H, Redelmeier TE. Skin Barrier. Principles of Percutaneous Absorption.
Karger, Basel, 1996.
4. Williams AC, Barry BW. Chemical penetration enhancement: possibilities and
problems. eds. In: Roberts MS Walters KA. In: Dermal Absorption and Toxicity
Assessment, Marcel Dekker, New York, 1998; 57:297–312.
5. Barry BW. Penetration enhancers. Mode of action in human skin. Pharmacol Skin
1987; 1:121–137.
6. Barry BW. Lipid-protein partition theory of skin penetration enhancement. J
Controlled Release 1991; 15:237–248.
7. Elias PM, Goerke J, Friend DS. Mammalian epidermal barrier layer lipids:
composition and influence on structure. J Invest Dermatol 1977; 69: 535–546.
8. Elias PM, Brown BE, Fritsch P, Goerke J, Gray GM, White RJ. Localization and
composition of lipids in neonatal mouse stratum granulosum and stratum corneum.
J Invest Dermatol 1979; 73:339–348.
9. Elias PM. Epidermal lipids, membranes and keratinization. Int J Dermatol 1981; 20:
1–19.
10. Elias PM. Lipids and the epidermal barrier. Arch Dermatol Res 1981; 270: 95–117.
11. Swartzendruber DC, Wertz PW, Kitko DJ, Madison KC, Downing DT Molecular
models of the intercellular lipid lamellae in mammalian stratum corneum. J Invest
Dermatol 1989; 92:251–257.
713.
13. Friberg SE. Micelles, microemulsions, liquid crystals, and the structure of stratum
corneum lipids. J Soc Cosmet Chem 1990; 41:155–171.
14. Forslind B. A domain mosaic model for the skin barrier. Acta Dermatol Venereol
(Stockh) 1994; 74:1–6.
15. Landmann L. The epidermal permeability barrier. Anat. Embryol 1988; 178: 1–13.
skin barrier. Acta Dermatol Venereol (Stockh) suppl 2000; 208:23–30.
17. Fartach M, Bassukas ID, Diepgen T. Structural relationship between epidermal
lipid lamellae, lamellar bodies and desmosomes in human epidermis: an
ultrastructural study. Br J Dermatol 1993; 128:1–9.
18. Elias PM, Holleran WM, Calhoun DQ, Brown BE, Behne M, Feingold KR.
Permeability barrier homeostasis: the role of lipid processing. In: M. Lodén and
H.I. Maibach, eds. Dry Skin and Moisturizers. Chemistry and Function, CRC
Press, Boca Raton, FL, 2000; 128:59–70.
19. Norlén L. Skin barrier formation: the membrane folding model. J Invest Dermatol
2001; 117:823–829.
20. Norlén L. Skin barrier structure and function: the single gel-phase model. J Invest
Dermatol 2001; 117:830–836.
21. Moloney SJ, Teal JJ. Alkane-induced edema formation and cutaneous barrier
dysfunction. Arch Dermatol Res 1988; 280:375–379.
22. Feingold KR, Elias PM. The environmental interface: regulation of permeability
barrier homeostasis. In: In: Lodén M and Maibach HI, eds. Dry Skin and
Moisturizers. Chemistry and Function, CRC Press, Boca Raton, FL, 2000; 280:45–
58.
23. Engström K, Husman K, Riihimäki V. Percutaneous absorption of m-xylene in
man. Int Arch Occup Environ Health 1977; 39:181–189.
24. Riihimäki V, Pfäffli P. Percutaneous absorption of solvent vapors in man. Scand J
Work Environ Health 1978; 4:73–85.
25. Linde YW. Dry skin in atopic dermatitis. Acta Dermal Venereol (Stockh) 1992
suppl; 9–13.
26. Imokawa G, Abe A, Jin K, Higaki Y, Kawashima M, Hidano A. Decreased level of
ceramides in stratum corneum of atopic dermatitis: an etiologic factor in atopic dry
skin?. J Invest Dermatol 1991; 96:523–526.
27. Imokawa G. Lipid abnormalities in atopic dermatitis. J Am Acad Dermatol 45
(supp1 1:2001) S29-S32.
28. Pilgram GS, Vissers DC, van der Meulen H, Pavel S, Lavrijsen SP, Bouwstra JA,
Koerten HK. Aberrant lipid organization in stratum corneum of patients with atopic
dermatitis and lamellar ichthyosis. J Invest Dermatol 2001; 117: 710–717.
29. Di Nardo A, Wertz P, Giannetti A, Seidenari S. Ceramide and cholesterol
composition of the skin of patients with atopic dermatitis. Acta Dermatol Venereol
(Stockh) 1998; 78:27–30.
30. Fartasch M, Bassukas ID, Diepgen TL. Disturbed extruding mechanism of lamellar
bodies in dry non-eczematous skin of atopics. Br J Dermatol 1992; 127:221–227.
31. Fartasch M. Epidermal barrier in disorders of the skin. Microsc Res Techniques
1997; 38:361–372.
32. Bleck O, Abeck D, Ring J, Hoppe U, Vietzke JP, Wolber R, Brandt O, Schreiner
V. Two ceramide subfractions detectable in Cer (AS) position by HPTLC in skin
surface lipids of nonlesional skin of atopic eczema. J Invest Dermatol 1999; 113:
894–900.
33. Yamamoto A, Serizawa S, Ito M, Sato Y. Stratum corneum lipid abnormalities in
34. Schafer L, Kragballe K. Abnormalities in epidermal lipid metabolism in patients
with atopic dermatitis. J Invest Dermatol 1991; 96:10–15.
35. Melnik B, Hollmann J, Hofmann U, Yuh MS, Plewig G. Lipid composition of
outer stratum corneum and nails in atopic and control subjects. Arch Dermatol Res
1990; 282:549–551.
36. Macheleidt O, Kaiser HW, Sandhoff K. Deficiency of epidermal protein-bound -
hydroxyceramides in atopic dermatitis. J Invest Dermatol 2002; 119:166–173.
37. Nassif A, Chan SC, Storres FJ, Hanifin JM. Abnormal skin irritancy in atopic
dermatitis and in atopy without dermatitis. Arch Dermatol 1994; 130:1402–1407.
38. Björnberg A Skin reactions to primary irritants in patients with hand eczema, Oscar
IsacsonTryckeri, Göteborg, 1968.
39. Löffler H, Effendy I. Skin susceptibility of atopic individuals. Contact Dermatitis
1999; 40:239–242.
40. Tupker RA, Pinnagoda J, Coenraads PJ, Nater JP. Susceptibility to irritants: role of
barrier function, skin dryness and history of atopic dermatitis. Br J Dermatol 1990;
123:199–205.
41. van der Valk GM, Nater JP, Bleumink E. Vulnerability of the skin to surfactants in
different groups of eczema patients and controls as measured by water vapour loss.
Clin Exp Dermatol 1985; 10:98–103.
42. Agner T. Susceptibility of atopic dermatitis patients to irritant dermatitis caused by
sodium lauryl sulphate. Acta Dermatol Venereol (Stockh) 1991; 71: 296–300.
43. Rystedt I. Factors influencing the occurrence of hand eczema in adults with a
history of atopic dermatitis in childhood. Contact Dermatitis 1985 185–191.
44. Nilsson E. Individual and environmental risk factors for hand eczema in hospital
workers. Acta Dermatol Venereol (Stockh) suppl 1986; 128:1–63.
45. Berardesca E, Fideli D, Borroni G, Rabbiosi G, Maibach HI. In vivo hydration and
water-retention capacity of stratum corneum in clinically uninvolved skin in atopic
and psoriatic patients. Acta Dermatol Venereol (Stockh) 1990; 70:400–404.
46. Werner Y, Lindberg M, Forslind B. The water-binding capacity of stratum corneum
in dry non-eczematous skin of atopic eczema. Acta Dermatol Venereol (Stockh)
1982; 62:334–337.
47. Melnik B, Hollmann J, Plewig G Decreased stratum corneum ceramides in atopic
individuals—a pathobiochemical factor in xerosis? Br J Dermatol 1988; 119:547–
548.
48. Menon GK, Elias PM. Ultrastructural localization of calcium in psoriatic and
normal human epidermis. Arch Dermatol 1991; 127:57–63.
49. Bouwstra J, Pilgram G, Gooris G, Koerten H, Ponec M. New aspects of the skin
barrier organization. Skin Pharmacol Appl Skin Physiol4 suppl 2001; 1:52–62.
50. Motta S, Sesana S, Monti M, Giuliani A, Caputo R. Interlamellar lipid differences
between normal and psoriatic stratum corneum. Acta Dermatol Venereol (Stockh)
suppl 1994; 186:131–132.
51. Hartop PJ, Allenby CF, Prottey C. Comparison of barrier function and lipids in
psoriasis and essential fatty acid–deficient rats. Clin Exp Dermatol 1978; 3:259–
267.
52. Gånemo A. (2002) Hereditary Ichthyosis: Causes, Skin Manifestations, Treatments
and Quality of Life. Uppsala; Acta Universitatis Upsaliensis 1125.
53. Zettersten E, Man MQ, Sato J, Denda M, Farrell A, Ghadially R, Williams ML,
Feingold KR, Elias PM. Recessive X-linked ichthyosis: role of cholesterol-sulfate
accumulation in the barrier abnormality. J Invest Dermatol 1998; 111:784–790.
54. Williams ML. Epidermal lipids and scaling diseases of the skin. Semin Dermatol
1992; 11:169–175.
55. Rehfeld SJ, Williams ML, Elias PM. Interactions of cholesterol and cholesterol
sulfate with free fatty acids: possible relevance for the pathogenesis of recessive X-
linked ichthyosis. Arch Dermatol Res 1986; 278:259–263.
56. Paige DG, Morse-Fisher N, Harper JI. Quantification of stratum corneum
ceramides and lipid envelope ceramides in the hereditary ichthyoses. Br J Dermatol
1994; 131:23–27.
57. Elias PM, Schmuth M, Uchida Y, Rice RH, Behne M, Crumrine D, Feingold KR,
Holleran WM, Pharm D. Basis for permeability barrier abnormality in lamellar
ichthyosis. Exp Dermatol 2002; 11:248–256.
58. Serizawa S, Nagai T, Ito M, Sato Y. Cholesterol sulphate levels in the hair and
nails of patients with recessive X-linked ichthyosis. Clin Exp Dermatol 1990; 15:
13–15.
59. Johansen JD, Ramsing D, Vejlsgaard G, Agner T. Skin barrier properties in
patients with recessive X-linked ichthyosis. Acta Dermatol Venereol (Stockh) 1995;
75:202–204.
60. Lodén M, Maibach HL, eds. Dry Skin and Moisturizers: Chemistry and Function,
Boca Raton, FL: CRC Press, 1999.
61. Hagstromer L, Nyren M, Emtestam L. Do urea and sodium chloride together
increase the efficacy of moisturisers for atopic dermatitis skin? A comparative,
double-blind and randomised study. Skin Pharmacol Appl Skin Physiol 2001; 14:
27–33.
62. Bettinger J, Gloor M, Peter C, Kleesz P, Fluhr J, Gehring W. Opposing effects of
glycerol on the protective function of the horny layer against irritants and on the
penetration of hexyl nicotinate. Dermatology 1988; 197:18–24.
63. Lodén M. Urea-containing moisturizers influence barrier properties of normal skin.
Arch Dermatol Res 1996; 288:103–107.
64. Lodén M. Barrier recovery and influence of irritant stimuli in skin treated with a
moisturizing cream. Contact Dermatitis 1997; 36:256–260.
65. Lodén M, Andersson AC. Effect of topically applied lipids on surfactant-irritated
skin. Br J Dermatol 1996; 134:215–220.
66. Tanojo H, Boelsma E, Junginger HE, Ponec M, Bodde HE. In vivo human skin
barrier modulation by topical application of fatty acids. Skin Pharmacol Appl Skin
Physiol, 1998; 11:87–97.
67. Lodén M, Andersson A-C, Lindberg M. Improvement in skin barrier function in
patients with atopic dermatitis after treatment with a moisturizing cream
(Canoderm®). Br J Dermatol 1999; 140:264–267.
68. Held E, Lund H, Agner T. Effect of different moisturizers on SLS-irritated human
skin. Contact Dermatitis 2001; 44:229–234.
69. Held E, Sveinsdottir S, Agner T Effect of long-term use of moisturizers on skin

hydration, barrier function and susceptibility to irritants, Acta Dermatol Venereol
(Stockh) 1999; 79:49–51.
70. Lippold BC, Hackemüller D. The influence of skin moisturizers on drug
penetration in vivo. Int J Pharm 1990; 61:205–211.
71. Duval C, Lindberg M, Boman A, Johnsson S, Edlund F, Lodén M. Differences
among moisturizers in affecting skin susceptibility to hexyl nicotinate, measured as
time to increase skin blood flow. Skin Res Techniques 2002; Accepted for
publication.
72. Wahlberg JE. Erythema-inducing effects of solvents following epicutaneous
administration to man-studied by laser Doppler flowmetry. Scand J Work Environ
Health 1984; 10:159–162.
73. Goldsmith LB, Friberg SE, Wahlberg JE. The effect of solvent extraction on the
lipids of the stratum corneum in relation to observed immediate whitening of the skin.
Contact Dermatitis 1988; 19:348–350.
74. Berenson GS, Burch GE. Studies of the diffusion of water through dead human
skin: the effect of different environmental states and of chemical alterations of the
epidermis. Am J Trop Med Hyg 1951; 31:842–853.
75. Malten KE, Spruit D, Boemars HGM, de Keizer MJM. Horny layer injury by
solvents. Berufsdermatosen l968; 16:135–147.
76. Allenby AC, Fletcher J, Schock C, Tees TFS. The effect of heat, pH and organic
solvents on the electrical impedance and permeability of excised human skin. Br J
Dermatol S 41969; 81:31–39.
77. Spruit D, Malten KE, Lipmann EWRM, The Poo Liang Horny layer injury by
solvents II Can injury of petroleum ether be diminished by pretreatment?
Berufsdermatosen 1970; 18:269–280.
78. Malten KE, den Arend J. Topical toxicity of various concentrations of DMSO
recorded with impedance measurements and water vapour loss measurements.
79. Boman A, Fernström P, Wahlberg, JE. Barriärskada i marsvinshud av organiska
lösningsmedel studerat med mätning av transepidermal vattenförlust. Slutrapport,
AMFO projekt 84–0294, Arbetsmiljöinstitutet. 1987 (in Swedish).
80. Imokawa G, Hattori M. A possible function of structural lipids in the water-holding
properties of the stratum corneum. J Invest Dermatol 1984; 84:282– 284.
81. Vinson LJ, Singer EJ, Koehler WR, Lehman MD, Masurat T. The nature of the
epidermal barrier and some factors influencing skin permeability. Toxicol Appl
Pharmacol 1965; 73:7–19.
82. Boman A, Wahlberg J E. Percutaneous absorption of 3 organic solvents in the
guinea pig (I). Effect of physical and chemical injury to the skin. Contact
Dermatitis 1989; 21:36–45.
83. Abrams K, Harvell JD, Shriner D, Wertz P, Maibach HI, Rehfeld SJ. Effect of
organic solvents on in vitro human skin water barrier function. J Invest Dermatol
1993; 101:609–613.
84. Menzel E. Skin delipidization and percutaneous absorption. In: In: Bronaugh RL
Maibach HI eds. Percutaneous Absorption, Mechanisms, Methodology, Drug
Delivery, Marcel Dekker, New York, 1985; 101:133.
85. Matoltsy AG, Downes AM, Sweeney TM. Studies of the epidermal water barrier.
II 1968; 101: Investigation of the chemical nature of the water barrier. J Invest
Dermatol 50:19–26.
86. Scheuplein R, Ross L. Effects of surfactants and solvents on the permeability of
epidermis. J Soc Cosmet Chem 1970; 21:853–873.
87. Riihimäki V. Percutaneous absorption of M-xylene from a mixture of M-xylene
and isobutyl alcohol in man. Scand J Work Environ Health 1979; 5:143–150.
88. Steele RH, Wilhelm DL. The inflammatory reaction in chemical injury. I. Increased
vascular permeability and erythema induced by various chemicals. Br J Exp Pathol
1966; 47:612–623.
89. Lupulescu AP, Birmingham DJ. Effect of protective agent against lipid-solvent-
induced damages. Arch Environ Health 1976; 31:29–32.
90. Hake CL, Stewart RD. Human exposure to tetrachloroethylene: inhalation and skin
contact. Environ Health Perspect 1977; 21:231–238.
91. Mahmoud G. Evaluation of the protective value of an antisolvent gel by laser Doppler
flowmetry and histology. Contact Dermatitis 1985; 13:14–19.
92. Jacobs GA, Castellazzi A, Dierickx PJ. Evaluation of a non-invasive human and an
in vitro cytotoxic method as alternatives to the skin irritation test on rabbit. Contact
Dermatitis 1989; 21:239–244.
93. Wahlberg JE. Edema-inducing effects of solvents following topical administration.
Dermatosen 1984; 32:91–94.
94. Rycroft RJG, Wilkinson JD. Irritants and sensitizers. In: Champion RH, Burton JL,
Ebling FJG, eds. Rook, Wilkinson, Ebling, Textbook of Dermatology, 5th ed.
Oxford: Blackwell Scientific Publications, 1992:718–754..
95. Frosch P.J. Clinical aspects of irritant contact dermatitis. In: RJG Rycroft, T
Menné, P J Frosch, J-P Lepoittevin, eds. Textbook of Contact Dermatitis, 3rd ed.
Berlin: Springer-Verlag, 2001:311–354.
96. Klauder JV, Brill FA. Correlation of boiling ranges of some petroleum solvents
with irritant action on skin. Arch Dermatol 1947; 56:197–215.
97. Suskind RR. Industrial and laboratory evaluation of a silicone protective cream. Ind
Hyg Occup Med 1954; 10:101–112.
98. Hunter GA. Chemical burns of the skin after contact with petrol. Br J Plast Surg
1968; 21:337–341.
99. Tagami H, Ogindo A. Kerosene dermatitis. Factors affecting skin irritability to
kerosene. Dermatologica 1973; 146:123–131.
100. Bauer M, Rabens SF. Trichloroethylene toxicity. Int J Dermatol 1977; 16: 113–116.
101. Shmunes E. Solvents and plasticizers. In: In: Adams RM 2nd ed. Occupational
Skin Disease, Saunders, Philadelphia, WB, 1990:439–461; 16:.
102. Dooms-Goossens AE, Debusscherre KM, Gevers DM, Dupré KM, Degreef HJ,
Loncke JP, Sauwaert JE. Contact dermatitis caused by airborne agents. J Am Acad
Dermatol 1986; 15:1–10.
103. Nethercott JR, Pierce JM, Likwornick G, Murray AH. Genital ulceration due to
Stoddard solvent. J Occup Med 1980; 22:549–552.
104. Koh D, Foulds IS, Aw TC. Dermatological hazards in electronics industry. Contact
Dermatitis 1990; 12:1–7.
105. Meding B, Swanbeck G Occupational hand eczema in an industrial city, Contact

Dermatitis 1990; 22:13–23.
106. Wall LM, Gebauer KA. Occupational skin disease inWestern Australia. Contact
Dermatitis 1991; 24:101–109.
10
Understanding the Irritative Reaction
Carolyn Willis
Amersham Hospital, Amersham, United Kingdom
Magnus Lindberg
Stockholm, Sweden
Contact dermatitis is a common problem in the general population and is the
most prevalent occupational disease in dermatology [1–4]. The cause is often
repeated exposure over time to a mild or low-grade irritant. Any skin site can be
affected, but the dermatitis is most frequently located on the hands or face, or in
intertriginous areas; it can be found in all age groups. The prevalence of hand
eczema, for example, is approximately 10% in the general population [5] and is
equally high in schoolchildren [6]. Irritant contact dermatitis (ICD) is considered
to be more common than allergic contact dermatitis (ACD). However,
combinations of the two are often seen in clinical practice, with constitutional
factors also playing a role.
Common causes of ICD are repeated contact with water, detergents, alkali and
other reactive chemicals, and cutting or cooling fluids. Handling foodstuffs or
plants may also give rise to ICD, as may mechanical friction and contact with
different forms of dust. Several studies have demonstrated a correlation between
the development of hand eczema and frequency of hand washing [7, 8].
Although the use of protective gloves is generally recommended, at times gloves
may exert a negative effect owing to their occlusive properties [9, 10].
An important and formerly poorly recognized form of skin irritation is that
resulting from the use of cosmetics and skin care products [11–13]. A recent
population-based study reported that as much as 53% of the general population
who perceived themselves as having sensitive skin had experienced unwanted
skin reactions following the use of such products [14]. With such high
prevalence rates, contact dermatitis can have farreaching socioeconomic
consequences, both for affected individuals and for society.
During the past two decades many studies have been performed on different
aspects of ICD, including skin barrier function. Most have focused on the
elicitation phase, although the period of repair and recovery is equally important
from the perspective of treatment and prevention.
216 WILLIS AND LINDBERG
1
DEFINITIONS AND CLINICAL ASPECTS
Irritant contact dermatitis has been defined as “a non-immunological local
inflammatory reaction characterised by erythema, oedema or corrosion following
single or repeated application of a chemical substance to an identical cutaneous
site” [15]. This definition can be expanded to include physical factors, such as
mechanical friction, dry dust, and cold [16]. In clinical practice, ICD can be
associated with a broad spectrum of signs and symptoms, and in the literature
several different clinical names can be found. However, there is general
agreement that ICD can be divided into four main clinical types: chemical burns,
irritant contact dermatitis (following a single exposure to a noxious factor),
chronic irritant dermatitis (following repeated exposures to noxious factors over
a period of time), and sensory irritation (stinging/smarting) [2, 17–19]. Clinical
signs encompass varying degrees and combinations of the classical signs of
inflammation in the skin, notably erythema, edema, papules, vesicles, scaling,
and fissures. Sterile pustules may occasionally be a feature of the reaction. The
predominant feature of chemical burns is erosion of the epidermis, while at the
opposite end of the spectrum, stinging/ smarting responses are purely sensory
and are not accompanied by visible skin changes [20–22]. For a significant
number of patients, the clinical presentation of ICD is indistinguishable from
that of ACD, making diagnosis difficult and unreliable [23].
2
SKIN REACTIVITY: FACTORS INFLUENCING SKIN
RESPONSE TO IRRITANTS
Irritant contact dermatitis has a multifactorial etiology, with clinical expression
and susceptibility to irritation being dependent on a plethora of factors [24–27].
In 1968, Alf Björnberg clearly demonstrated the wide variation in interindividual
responses to one and the same irritant, and to different irritants [28]. This finding
has important implications for the design of investigative studies, particularly
with respect to sample size. A list of factors influencing the skin response to
irritants with references to published work is given in Table 1. In considering the
results of these
TABLE 1 Factors Influencing Skin Reactivity to Irritants

Factor Comments and references
Chemical properties of the applied Determines the skin penetration and
substance and its vehicle noxious capacity [130]
Concentration of the substance, frequency Determines the skin penetration and
of application, exposed skin area noxious capacity [130]
Occlusion of the skin Increases hydration of the skin and
penetration [131–133]
UNDERSTANDING THE IRRITATIVE REACTION 217
Factor Comments and references

Age See [134, 135]
Sex Possible differences in skin reactivity due
to gender not completely resolved; most
studies indicate no gender differences
[136, 137]
Ethnic background Possible differences in skin reactivity due
to ethnic background not resolved [29, 30]
Anatomical location See [138] and [139]
Diurnal variations Possible variations in barrier function over
24 hr period [141]
Seasonal variations Increased susceptibility during winter
season
Skin areas with healed ICD See [31] and [142]
Presence of atopic dermatitis Increased reactivity, impaired diffusion
barrier [33, 142–145]
Presence of other Seborrheic dermatitis [147],
forms of dermatitis exogenous eczema [148],
subclinical irritation [149]
References provided for suggested reading.
studies, however, it is important to take into account the robustness of the

experimental design. Small sample sizes, which will inevitably magnify the
effects of interindividual variation, may well lead to unreliable assertions
[29–31].
It has been suggested that the quality of the skin barrier, as indicated by the
measurement of transepidermal water loss (TEWL), is a good predictor of
susceptibility to developing ICD [32–36]. This raises the possibility that
variation in individual barrier function contributes significantly to observed
variability in skin reactivity. This is particularly likely at sites previously affected
with ICD [31], and has been indicated as a factor in individuals exhibiting
sensitive skin [22]. Furthermore, those affected by atopic dermatitis, who have a
well-established increased risk of developing ICD [1, 37], are known to have an
impaired barrier function, as shown by increased TEWL. This has been attributed
to an inherent reduction in the ceramide fraction of the intercellular lipids
[38–40]. In support of this, under experimental settings, correlation has been
shown between increased sensitivity to sodium lauryl sulfate and the ceramide
content of the skin [41].
3
SKIN BARRIER BIOLOGY AND IRRITANT CONTACT
DERMATITIS
Preserving the internal milieu of the body and maintaining control of body water
are among the most crucial functions of the skin barrier. The water diffusion
barrier, located in the stratum corneum, is composed of proteinrich corneocytes
embedded in a lipid-rich intercellular space. The structural and functional
organization of the stratum corneum components, as well as the mechanisms
controlling the function and the maintenance of this diffusion barrier, have been
extensively investigated since the late 1970s [42–47]. Today, it is clear that any
perturbation of the barrier function immediately initiates processes designed to
repair the disturbance [48]. These include increased DNA synthesis in
keratinocytes and upregulation of the local production and processing of barrier
lipids. It appears that one of the most crucial steps in controlling the response to
barrier perturbation is the distribution and redistribution of calcium in the
epidermis [49–51]. Alterations in the transepidermal gradient of other
physiological important elements are also likely to play an important role [52–54].
The signal to start the response appears to be a change in TEWL [55].
Perturbation of the barrier is also associated with the induction of an
inflammatory response. Epidermal keratinocytes not only produce and maintain
stratum corneum, but also have the capacity to produce inflammatory mediators
and express adhesion molecules on exposure to noxious stimuli [56–58]. Today,
it is considered that the epidermal response to environmental factors is complex,
with nonspecific mechanisms acting together with more specific immune
reactions [59–62]. Following topical exposure to irritants, several interactions
may be hypothesized.
1. A direct effect on the stratum corneum, resulting in an impaired barrier and

an increased risk of penetration of irritants and allergens, together with the
initiation of barrier repair.
2. Penetration into the epidermis below the level of the stratum corneum, with
direct effects on the keratinocytes leading to (a) altered differentiation and
barrier production that result in an impaired barrier and/or (b) direct
cytotoxic effects initiating the release of inflammatory mediators and the
upregulation of adhesion molecules.
3. Penetration through the epidermis, with possible effects on dermal
components and/or passage into the circulation.
4
TECHNIQUES FOR EVALUATING SKIN IRRITATION
The skin is stratified in terms of both structure and function. It is a thin, living
tissue and presents a large surface area that can interact with and be influenced
by environmental factors. Taking into consideration the regulation and control of

the barrier function, the different defense mechanisms, such as nonspecific
inflammation and immunological events, and the interindividual differences in
irritant responses, it is clear that different investigative techniques must be
applied in combination to understand the complexity of ICD. Some of the main
techniques that have been used for studies on ICD are listed in Table 2. Selected
references are given for further reading.
For studies of cellular biology, morphology, inflammation, and immunology,
established research techniques have been used and adapted to the specific
demands of the skin. Skin, being both thin and stratified, presents some technical
problems in preparing tissue for analysis. For example, epidermis is composed of
keratinocytes in different stages of differentiation, mixed with populations of
Langerhans cells and melanocytes. The varied structure of the skin also causes
problems in the interpretation of studies of physiological events occurring at the
cellular level. However, some investigative techniques (e.g., X-ray microanalysis
using particle probes, non invasive bioengineering techniques) have been found
to be well suited for studies on ICD. The use of X-ray microanalysis with
particle probes allows one to determine the levels of physiologically interesting
elements, such as sodium and
TABLE 2 Major Techniques Used in the Study of Irritant Contact Dermatitis

Question under study Methodology
Incidence and prevalence Clinical Epidemiological studies [1, 4]
characterization Morphology, Observational clinical studies [37] Light
ultrastructure and transmission electron microscopy (for
references, see Sec. 5.1.1)
Proliferative response of keratinocytes, Histochemical and immunohisto-chemical
immune and inflammatory responses (e.g., techniques (light and electron
leukocytes, antigen-presenting cells, microscopic), molecular biology
inflammatory mediators, adhesion techniques, different experimental models
molecules) (for references, see Sec. 5.1)
TEWL, water content, blood flow, surface Noninvasive bioengineering techniques:
structure, erythema, mechanical properties evaporimetry, impedance and capacitance
techniques, laser Doppler measurements,
erythema readings, replica techniques,
ultrasound, techniques to measure friction
and elasticity [67–70, 150]
Elements of physiological interest (e.g., Particle probe techniques: proton induced
sodium, phosphorus, potassium, calcium, X-ray emission (PIXE), energy dispersive
trace elements, and metals, e.g., nickel) X-ray microanalysis (EDX) [53, 54, 63, 64]
calcium, in specimens taken from ICD sites, and to relate these to data collected
by means of other techniques [53, 54, 63–66].
Noninvasive bioengineering methods have also been extensively used in
studies on contact dermatitis (Table 2) to measure such parameters as TEWL,
hydration, and erythema. Their application in dermatological research has been

well described [67–70]. A major advantage of these latter techniques is the ease
with which repeat measurements can be taken at the same site over a long time
period without interference to the skin. It has to be remembered, however, that
the parameters measured are the result of several integrated events in the skin. To
obtain a correct picture of a reaction and its dynamics, it is thus necessary to
combine several techniques. To standardize results among investigators,
guidelines have been published for evaporimetry [71], laser Doppler flowmetry
[72] and erythema measurements [73].
5
EFFECTS OF APPLICATION OF AND EXPOSURE TO
IRRITANTS
The effects on the skin of topically applied irritants will depend on a range of
factors, not least the circumstances of exposure. Sections 5.1 and 5.2 consider the
skin changes that occur following single application of irritant chemicals and
repeated exposure, respectively. It is perhaps fair to say that for both acute and
chronic ICD, information is most limited with regard to the recovery phase. In
daily life, ICD is most likely to arise from repeated exposure to weak irritants.
This is perhaps to be expected when one considers that impairment of barrier
function has been shown to persist for a period of several weeks following
application of even mild irritants, thereby rendering the skin more susceptible to
the effects of ongoing irritant exposure.
5.1
Single Exposure to Irritants
Single exposure to irritants, most often carried out experimentally by way of
occlusive patch testing, results in pathophysiological changes to the skin, the
nature and severity of which are influenced by the physicochemical properties of
the chemical, the concentration, dose and duration of application, and the
inherent susceptibility of the individual. Many different chemicals are capable of
inducing inflammation when applied at sufficiently high concentration for
sufficient time, and there is increasing evidence that they do so, particularly in
the early stages, by different mechanisms [74]. This is reflected first in the
clinical appearance of the patch test reactions induced, most notably after 48 hr of
exposure. Erythema is characteristic of virtually all reactions, but it shows subtle
variations with regard to hue and color saturation, perhaps depending on whether
increased blood flow through superficial blood vessels has arisen or whether
stasis and congestion exists [75]. The extent of edema is also variable, and
surprisingly unpredictable. In a dose-response study carried out with sodium
lauryl sulfate (SLS), edema occurred in several individuals, not in the most
erythematous reaction to the highest concentration (5%), as might be expected,
but in the responses to two lower concentrations (2.5 and 1.25%)(unpublished

findings). Another hallmark of irritant reactions, namely surface changes, also
shows considerable variability among irritants, both qualitative and quantitative,
during the acute inflammatory phase. The long-chain fatty acid nonanoic acid, for
example, induces pronounced wrinkling of the skin after 48 hr, while dithranol,
an antipsoriatic agent, causes little or no surface change. Among the factors
likely to contribute to these differences are the varying degrees to which irritants
exert such biochemical actions as the removal of stratum corneum lipids and
naturally occuring hygroscopic materials, the denaturation of keratin, and the
extraction of proteins and amino acids. All these will impact skin barrier
function, leading to visible clinical changes and/or measurable increases in
transepidermal water loss, generally concomitant with decreases in hydration
levels.
The heterogeneity of the responses to irritants differing in their molecular
structure is further reflected in the histopathological changes exhibited in the
viable epidermis and in the dermis, and in many of the early
immunopathological events that follow single patch test exposure. Initial damage
to deeper layers of the skin takes a number of different forms, depending on the
mechanisms of action of the chemical. Cellular membrane disruption or
perturbation may occur, particularly in the case of surfactants, leading at an early
stage to the direct release of active preformed proinflammatory cytokines, such
as interleukin 1 (IL-1 ), and of tissue-destructive enzymes. Direct effects by
selected irritants on dermal vessels have also been reported. Together, these and
other pathophysiological events initiate cascades of inflammatory mediator
production and release, probably to a large extent common to all irritant
reactions, which regulate the eventual repair and regeneration of affected skin.
Sections 5.1.1 to 5.1.8 present details of some of the major cellular features of
single exposure to irritants.
5.1.1
Histopathology and Ultrastructure
Over the years there have been many studies of the morphology of irritant patch
test reactions, and a wide variety of histological features have been described.
Table 3 provides a summary of the predominant changes seen in the epidermis
and dermis, the occurrence and severity of which are
TABLE 3 Histopathological Changes in the Epidermis and Dermis Following a Single

Exposure to Irritant Chemicals
Epidermis Dermis
Spongiosis Edema
Vesiculation/bullae Collagen disruption/degeneration
Intracytoplasmic edema/vacuolation Capillary dilatation
Epidermis Dermis
Nuclear vacuolation/pyknosis Hyperemia
Necrosis Mast cell degranulation
Parakeratosis
Acantholysis
Hydropic degeneration/swelling
Dyskeratosis
Epidermal/dermal separation
Source: Refs. 151–159.
influenced by the chemical nature and concentration of the irritant applied, the
duration of exposure, the severity of the ensuing response, and the time of
sampling. Variability related to the irritant is exemplified by several studies
specifically aimed at comparing the effects on the skin of irritants with different
physicochemical properties. In an electron microscopic study of the stratum
corneum, utilizing ruthenium tetroxide as a secondary fixative, Fartasch and
coworkers elegantly demonstrated that topical exposure to SLS leads to damage
to the nucleated cells of the epidermis [76], while acetone induces disruption at
all levels of the stratum corneum. Similarly, Nagao et al. described significant
ultrastructural differences in the responses elicited by brief exposure to sodium
hydroxide and hydrochloric acid [77]. The former dissolved the contents of
horny cells and disrupted tonofilament-desmosome complexes, changes that
were not apparent with the latter.
Using 1 µm, plastic sections stained with toluidine blue in combination with
electron microscopy, Willis et al. [74] also demonstrated the principle that single
exposure to structurally unrelated irritants leads to variations in morphology that
are attributable to varying mechanisms of cellular damage. For reactions of
similar clinical intensity, changes to keratinocytes, in particular, showed
specificity according to the irritant applied. The anionic detergent SLS produced
primarily parakeratosis after exposure for 48 hr (Fig. 1), contrasting with the
cationic detergent benzalkonium chloride, which gave rise to marked spongiosis
and exocytosis, with focal areas of necrosis (Fig. 2). Another irritant with
detergent properties, nonanoic acid, gave yet another pattern of change, that of
tongues of dyskeratotic keratinocytes extending down from the stratum
granulosum into the stratum spinosum (Fig. 3).
Immunopathological evaluation of the leukocytes that infiltrate ICD patch test
sites has also revealed another aspect of the response that is, to a degree, irritant
dependent. In qualitative terms, there is commonality in the phenotypes of the
inflammatory cells present in both the epidermis and dermis: CD4+ cells
predominate, with CD8+, CD1a+, macrophages, and, in severe reactions,
neutrophils also being present, the vast majority of the cells expressing HLA-
DR. Quantitatively, however, there are significant differences among irritants,
particularly in the epidermis. In reactions of similar clinical intensity, croton oil,

for example, produces a marked exocytosis, while nonanoic acid induces very
little in the way of epidermal infiltration (Fig.4) [78].
5.1.2
Epidermal Elemental Content
The development of particle probes has made it possible to determine the levels
of physiologically interesting elements in freeze-dried sections of skin and also
in frozen hydrated specimens [53, 54, 63, 64, 79–81]. This, in turn, reveals
information on the functional state of the cells (e.g., cell injury, cell death, cell
proliferation), since changes in the ratio of sodium to potassium and in the
cellular concentrations of magnesium, phosphorus, potassium, and calcium are
known to occur during these events. Several studies on irritant contact dermatitis
have been carried out by means of energy-dispensive X-ray microanalysis
(EDX) with the electron probe [65, 66]. Following occlusive application of SLS
and nonanoic acid for 24 hr, changes to keratinocytes compatible with cell
membrane injury are detectable at 48 hr with both substances. However, in
biopsy samples taken prior to 24 hr, there are variations in the response to the
two irritants, detectable as differences in the sodium/potassium ratios [66]. These
results are in accordance with the suggestion that chemically different irritants
induce different responses in the skin. Samples removed later than 48 hr after
SLS and nonanoic acid application are compatible with a stimulated keratinocyte
population [65].
5.1.3
Langerhans Cells
Although not thought to play the pivotal role in irritant reactions that they have
in allergic contact dermatitis, Langerhans cells (LC) do nevertheless exhibit
significant changes in morphology and epidermal density following single
exposure to irritants (Fig. 5). Electron microscopy of patch test reactions has
revealed a variety of cellular changes to LC, some indicative of activation
(Fig. 6), others of degeneration (Fig. 7, Table 4). Where direct disruption to cell
membranes and organelles occurs, it is not unreasonable to assume that this
would result in the release of preformed cytokines, such as IL-1 , in a similar
manner to that demonstrated for keratinocytes. Table 5 presents the results from
a number of studies in which Langerhans cell numbers in the epidermis were
quantified during the evolution of irritant reactions; it is immediately apparent
that there is little consensus. Differences in experimental design and methods of
quantitation are probably responsible to a large extent for the discrepancies shown
[82]. Where significant decreases do occur, the irritants applied may have caused
irreversible cellular damage to LC, leading to their rapid removal from the
epidermis. Alternatively, the reduction in numbers may represent a more active
FIGURE 1 Transmission electron micrographs showing the appearance of the

parakeratotic upper epidermis typical of 48 hr patch test reactions to SLS (5%). Cells
immediately beneath the stratum corneum contain homogeneously staining, condensed
chromatin within the nuclei (A). The darkly stained parakeratotic cells beneath have dense
osmiophilic cytoplasm containing numerous lipid droplets and membrane-bound vesicles,
with no keratohyalin granules (B). An abrupt transition to paler keratinocytes with a more
typical appearance then occurs.
process, resulting from the local release of IL-1 and tumor necrosis factor
(TNF- ), both of which are known to promote the migration of LC away from
FIGURE 2 Skin biopsy sample taken from an individual patch-tested for 48 hr with
benzalkonium chloride (0.5%). The transmission electron micrograph shows spongiosis
or intracellular edema in the lower epidermis accompanied by infiltration of mononuclear
leukocytes. Occasional areas of necrosis are seen in the upper epidermis (not shown).
the epidermis to the local draining lymph nodes, in a dose-dependent manner
[83, 84].
FIGURE 3 Patch testing for 48 hr with nonanoic acid (80%) produces tongues of
dyskeratotic keratinocytes extending downward from the upper epidermis. The
transmission electron micrograph illustrates the dense, wavy aggregates of osmiophilic
keratin filaments present within these cells.
5.1.4
Cytokine Release
The cytokines alluded to in the preceding section are members of a large family
of relatively recently identified inflammatory mediators that exert potent
biological effects on the growth, differentiation, and function of cells of most
types [85]. Most cytokines exhibit more than one function, and many
FIGURE 4 Photomicrographs illustrating the quantitative variation in epidermal

leukocyte infiltration induced by different irritants. In these 48 hr patch test responses of
similar clinical intensity, large numbers of exocytotic CD11a+ leukocytes are present in
reactions to croton oil (0.8%) (A), contrasting with the absence of epidermal infiltration in
nonanoic acid (80%) reactions (B) (original magnification ×200) (immunoperoxidase
labeling of frozen sections).
TABLE 4 Ultrastructural Changes Exhibited by Langerhans Cells Following a Single
Patch Test Exposure to Irritants
Activation Damage/Degeneration
Distended endoplasmic reticulum Vesiculation
Increased numbers of mitochondria Disruption to cell membranes
FIGURE 5 Immunoperoxidase labeling of normal skin (A) with an anti-CD1a+ antibody,

demonstrating the typical density and dendritic appearance of epidermal Langerhans cells.
Reductions in number and a loss of dendrites frequently occur in acute ICD, as illustrated
by this 48 hr patch test reaction to nonanoic acid (80%) (B) (original magnification
×200).
Activation Damage/Degeneration
Increased frequency of Birbeck granules Condensed nuclear heterochromatin
Enlarged nuclei Lipid accumulation
Source: Refs. 76, 160–162.
FIGURE 6 A metabolically active Langerhans cell in apposition to a lymphocyte in a 48

hr reaction to benzalkonium chloride (0.5%) (original magnification ×6200).
TABLE 5 Changes in Langerhans Cell Density Following a Single Exposure to Selected
Irritants
Density Irritant (%) Time Ref.
Decreased SLS (0.25, 0.5, 1) 48 hr [163]
SLS (0.5, 1) 96 hr [164]
SLS (10) 72 hr [165]
SLS (10) 2–14 days [166]
SLS (10) 1–8 days [167]
Dithranol (0.1) 8, 48 hr [168]
Dithranol (0.2) 48 hr [169]
Croton oil (1) 1–8 days [167]
Nonanoic acid (80) 24, 48 hr [95]
Nonanoic acid (80) 48 hr [76]
FIGURE 7 A damaged Langerhans cell displaying disrupted organelles and membranes,

following 48 hr patch testing with benzalkonium chloride (0.5%) (original magnification
×6700).

Unchanged SLS (0.5) 6, 24 hr [170]
SLS (5) 48 hr [76]
SLS (5) 4–72 hr [171]
SLS (10) 1 and 21 days [166]
SLS (10) 28 days [167]

Dithranol (0.2) 24, 48 hr [172]
Croton oil (0.8) 48 hr [76]
Croton oil (1) 4–72 hr [171]
Increased BC 48 hr [76]
SLS (0.5) 48, 96 hr [170]
SLS (2.5) 6–72 hr [173]
SLS (3) 4–5 weeks [174]
SLS (4) 48 hr [95]
SLS, sodium lauryl sulfate; BC, benzalkonium chloride.
show pleiotropic, overlapping activities. Complex interactions frequently take

place between them, such that cell functions may be affected in a synergistic,
additive, or antagonistic manner.
There is considerable evidence that cytokines play a significant role in the
pathogenesis of many, if not all, skin diseases, and, furthermore, that the
distinctive patterns of inflammation exhibited during the course of different
conditions are largely attributable to the patterns of cytokine release [86].
Importantly, cytokines are released not only by infiltrating leukocytes, but also
by resident skin cells, including keratinocytes, Langerhans cells, and mast cells,
all of which are known to participate in the responses to irritant chemicals [57].
As mentioned earlier, damage to human epidermal cells leads to the direct
release of IL-1 , which, in addition to its effects on LC migration, stimulates the
further release of IL-1 and the synthesis and release of other cytokines. These
include chemotactic cytokines (chemokines), such as IL-8, and growth-
promoting cytokines, such as IL-6 and granulocyte/ macrophage colony-
stimulating factor (GM-CSF) [87].
Our knowledge of the full spectrum and time course of cytokine and
chemokine release following single irritant exposure in vivo in man is very
limited. Table 6 summarizes the few studies that have been conducted to date, all
but one utilizing SLS alone to elicit the inflammatory response. In addition to
IL-1, another primary proinflammatory cytokine, TNF- , has
TABLE 6 Cyokines and Chemokines Detected Following Irritant Exposure in Man

Experimental design Cytokines [time detected (hr)]
SLS and NAA: patch tests, analysis of IL-1 , IL-1 , IL-8 (4, 8, 24), GM-CSF (4,
mRNA in shave biopsies by RT-PCR [66] 8, 24; SLS only), IL-6 (4, 8, 24; 3/3 NAA
subjects; 1 /3 SLS subjects)
SLS: patch tests, immuno-histochemical IL-1 , TNF- , IL-4, IL-6, IL-10 (6, 24,
analysis of biopsies [175] 72), IL-2, IFN- (24, 72)
SLS: patch tests, immuno-cytochemical IL-6 (48)
analysis of biopsies [176]
SLS: patch tests, ribonuclease protection IL-8 (72)
assay of tape strips [177]
Experimental design Cytokines [time detected (hr)]

SLS: collection of skin lymph draining TNF- , IL-6 (both “early phase”) IL-1 ,
site of patch test [178] GM-CSF (both “late phase”)
SLS, sodium lauryl sulfate; NAA, nonanoic acid; IL, interlenkin; IFN, interferon; GM-
CSF, granulocyte-macrophage colony-stimulating factor.
been detected on a number of occasions at patch test sites. This cytokine is stored
in dermal mast cells and may thus be directly released upon damage to mast cell
membranes [88]. However, keratinocytes and Langerhans cells are also potential
sources of TNF- , both being capable of its synthesis following appropriate
stimulation [89]. An investigation by Grängsjö et al. has suggested that, in
common with other aspects of the pathogenesis of ICD, there is heterogeneity
among irritants [66]. These investigators used a reverse transcriptase polymerase
chain reaction (RT-PCR) technique to assess shave biopsies taken from patch
tests of similar clinical intensity. They found that SLS gave a cytokine profile
slightly different from that of nonanoic acid.
5.1.5
Eicosanoid Release
Functioning in conjunction with cytokines to induce the inflammatory changes
observed in skin exposed to irritants, arachidonic acid metabolites also show
variation in their profile of release according to the irritant applied [90]. Table 7
provides a summary of the proinflammatory lipid mediators that have been
identified in irritant reactions, many of which are thought to be derived from
epidermal keratinocytes, particularly in the early stages of the irritative response.
TABLE 7 Eicosanoids Detected Following a Single Application of Irritant

Experimental design Eicosanoid
Four surfactants: suction blisters overlying SLS: PGE2/D2, 5-HETE BC: PGF2 , 6-
24 hr patch tests [179] keto-PGF1 , 12-HETE, LTB4
Tween 80: AA, 5-HETE, 12-HETE, 15-
HETE
TEA: no significant release
BC: suction blisters overlying 24 hr patch PGE1
tests [180]
Dithranol: 12 hr exposure, collection of PGE2 at 24, 48 hr 12-HETE at 72 hr
suction bullae fluid at 12, 24, 48, 72 hr
[181]
BC: 24 hr exposure, followed by perfusion PGE/PGF
for 60–90 min [182]
SLS, sodium lauryl sulfate; BC, benzalkonium chloride; PGE, PGF, prostaglandins E, F;
HETE; hydroxyeicosatetraenoic acid; LTB, leukotriene B; AA, arachidonic
acid; TEA, triethanolamine.
5.1.6
Adhesion Molecules
Among the many functions of cytokines is the modulation of expression of
adhesion molecules. These cell surface glycoproteins are produced by a variety of
cell types, including keratinocytes, and they play an essential role in the
trafficking of leukocytes from peripheral blood vessels to the epidermis and dermis
[91]. Although relatively few adhesion molecules have been studied in human
irritant reactions in vivo, it is likely that a commonality in expression will be
found in inflammatory skin conditions that exhibit similar patterns of leukocyte
infiltration. A study by Friedmann et al. demonstrated the upregulation of E-
selectin and vascular cell adhesion molecule-1, on dermal microvascular
endothelial cells within 2 hr of irritant application, followed by enhanced
expression of intercellular adhesion molecule-1 (ICAM-1), within 8 hr [92]. The
latter serves as one ligand for lymphocyte function-associated antigen-1 (LFA-1)
constitutively expressed by leukocytes [93], and has been demonstrated, not only
on endothelial cells, but also on the surface of keratinocytes in irritant reactions
by some, but not all, investigators (Fig. 8) [94, 95, 96, 97]. The discrepancy in
findings may be accounted for by the variability in cellular response between
different irritants and between reactions of differing intensity. Expression of
ICAM-1 by keratinocytes shows a very close spatial relationship with exocytotic
leukocytes in the epidermis [96]; therefore in patch tests reactions with limited
epidermal infiltration, ICAM-1 expression will be correspondingly minimal. The
upregulation of ICAM-1 on keratinocytes in ICD is, arguably, to be expected,
given the likely participation of interferon gamma (IFN- ) and TNF- in the
reactions, both of which are have been shown to induce ICAM-1 expression [98, 99]
.
5.1.7
Oxidative Stress
Oxidative stress is increasingly being seen as a contributory factor in inflammatory
skin conditions, including ICD. Excessive production of reactive oxygen species
(ROS), inadequately quenched by antioxidant mechanisms, results in
peroxidation of cell membrane lipids and damage to proteins and DNA [100].
There is also evidence that ROS exert immunomodulatory effects, such as the
enhancement of ICAM-1 expression by keratinocytes [101] and the regulation of
cytokine release [102, 103].
Some chemicals, including most notably dithranol, are capable of generating
ROS directly [104, 105]. In the main, however, the primary source of ROS, such
as superoxide anion and hydrogen peroxide, is infiltrating leukocytes, which are,
of course, present in abundance in the majority of irritant reactions.
Support for oxidative stress as a mechanism in acute ICD in man has come
from several studies that have examined changes in the levels of the skin’s innate
FIGURE 8 Immunoperoxidase-labeled serial sections taken from a biopsy sample of a 48

hr patch test reaction to benzalkonium chloride (0.5%). The close spatial relationship
between ICAM-1+ basal keratinocytes (A) and infiltrating LFA-1+ leukocytes (B) is seen
(original magnification ×200).
antioxidant defense enzymes by quantitative immunocyto-chemistry. Reduced

levels of copper/zinc superoxide dismutase (Cu, Zn-SOD) and glutathione S-
transferase were seen in the epidermis following patch testing with both
dithranol and SLS (Fig. 9, 10) [106, 107]. The hypothesis is further supported by
evidence of inhibition of inflammation following the application of ROS
inhibitors/scavengers. Superoxide dismutase itself has been shown to reduce the
erythema induced by dithranol and laurylsarcosine, as have other naturally
FIGURE 9 Graph showing the changes in the levels of Cu, Zn-superoxide dismutase
(SOD) in human epidermis following 48 hr patch-testing with dithranol (0.2%) (solid,
dithranol; diagonal line, white soft paraffin; dotted, normal skin) (mean+S.D.).
Significant reductions were seen at each of the three time points examined (quantitative
immunocytochemical technique).
occurring antioxidants, such as catalase and the redox couple dihydrolipoate-
lipoate [108, 109].
5.1.8
Epidermal Proliferation, Keratinization, and Differentiation
The acute inflammation induced by single exposure to chemical irritants leads to
the eventual repair and rejuvenation of the affected skin, and the restoration of
barrier function. Irrespective of the irritant applied, an essential component of
this process is a burst of proliferative activity by keratinocytes, detectable by
techniques such as Ki-67 expression and ornithine decarboxylase induction [65, 110
]. In human patch test reactions, this increase in the rate of division generally
starts to occur at around 48 to 96 hr [111, 112]. There appears to be a degree of
variability according to the irritant applied (Fig. 11, 12). Detergents, in
particular, are potent stimulators of proliferation, both in in vivo models of acute
irritation and in models of chronic irritation [113–115].
Closely associated with these enhanced rates of keratinocyte division are
alterations in the processes of keratinization and differentiation. Premature
expression of involucrin and transglutaminase occurs [110, 112, 114], with
upregulation of keratin-16 and keratin-17 expression (Fig. 13) [56, 106, 112, 116].
FIGURE 10 Graph showing the changes in the epidermal levels of the anti-oxidant
enzyme glutathione S-transferase (GST) alpha, following patch-testing with SLS (5%)
(solid SLS; diagonal line water, dotted normal skin) (mean+S.D.). Statlstically significant
reductions are seen after 48 h and 96 h (quantitative immunocytochemical technique).
5.2
Repeated Exposure to Irritants
Studies on the effects of repeated application of irritants to human skin have
almost exclusively utilized noninvasive bioengineering techniques.
Transepidermal water loss, water content of stratum corneum, and dermal blood
flow have been the major parameters used to investigate barrier function and the
inflammatory response. Repeated exposure to subclinical doses of detergents
increases the reactivity to subsequent exposure to irritants, while elicitation of an
irritant reaction is enhanced at sites of repeatedly induced irritant reactions
[117]. The possibility of hyporeactivity following the recovery of irritant
reactions has been suggested, as has the possibility of delayed forms of irritant
reactions [2, 19, 23]. Today, most investigators working in this area focus on the
development of experimental models that can be used to evaluate the efficacy of
barrier creams [117– 121]. It has been shown that there is variation in the chronic
irritant response depending on the choice of applied substance. The effects of
repeated skin exposure to combinations of different irritants has been
investigated to a minor extent [122–125]. Experiments using combinations of
detergents and solvents, demonstrated that the effects were more complex than
simply additive.
Undoubtedly ICD has a multifactorial etiology, but our knowledge of all the
relevant factors, particularly as they exist in normal everyday life, is limited. The
effects of physical stimuli on chronic ICD, for example, have not been studied in
FIGURE 11 Immunoperoxidase staining of 48 hr patch test reactions, using the antibody,

Ki-67, which labels proliferating cells. Large numbers of darkly stained, Ki-67+ basal
keratinocytes are typically seen in responses to SLS (5%) (A), contrasting sharply with
the paucity of dividing cells in dithranol (0.2%) reactions (B) (original magnification
×200).
depth. However, there are suggestions that temperature influences the response
to detergents [126–128] and that irritancy due to sodium lauryl sulfate enhances
the response to thermal stimuli [129].
FIGURE 12 Graph showing the variability in the numbers of proliferating Ki67+

keratinocytes in the epidermis following 48 hr patch testing with a range of irritants.
Statistical analysis showed the variability to be irritant-related rather than being
dependent on the intensity of reaction. BC, benzalkonium chloride; SLS, sodium lauryl
sulfate; CO, croton oil; NAA, nonanaoic acid; PG, propylene glycol; Dith, dithranol.
6
CONCLUSIONS
Irritant contact dermatitis remains a major problem in dermatology. Most often
resulting from repeated exposure to noxious factors in the environment, it can
deleteriously affect many areas of life, not least the ability to carry out one’s
chosen occupation. As challenges for the future, we need to increase our
knowledge of the mechanisms of ICD, particularly with respect to repeated
exposure to irritants and to combinations of irritants. We also need an increased
understanding of the mechanisms that influence and control the recovery phase of
irritant skin reactions.
REFERENCES
1. Diepgen T.L., Coenraads P.J. The epidemiology of occupational dermatitis. In:
L.Kanerva, P.Elsner, J.E.Wahlberg, H.I.Maibach, eds. Handbook of Occupational
Dermatology, Berlin, Springer-Verlag, 2000; 3–16.
2. Frosch P.J. Clinical aspects of irritant contact dermatitis. In: R.J.G. Rycroft,
T.Menné, P.J. Frosch, J.-P. Lepoittevin, eds. Textbook of Contact Dermatitis ed
3.Berlin: Springer-Verlag, 2001; 79:311–354.
3. Halkier-Sørensen L.; Haugaard Peterson B., Thostrup-Pedersen K. Epidemiology
of occupational skin diseases in Denmark: notification, recognition and
FIGURE 13 Keratin-16 (A) and keratin-17 (B) expression on serial frozen sections of a
biopsy taken from an individual patch-tested for 48 hr with SLS (5%). Expression of both
keratins is epibasal and somewhat patchy along the length of the epidermis. (Original
magnification ×200; no hematoxylin counterstain.)
compensation. In: P.G.M. van der Valk, H.I. Maibach, eds. The Irritant Contact
Dermatitis Syndrome, Boca Raton, FL, CRC Press, 1996; 23–52.
4. Shenefelt P.D. Epidemiology of irritant contact dermatitis. In: P.G.M.van der Valk,
H.I.Maibach, eds. The Irritant Contact Dermatitis Syndrome, Boca Raton, FL, CRC
Press, 1996; 21–22.
5. Meding B., Swanbeck G. Epidemiology of different types of hand eczema in an
industrial city. Acta Dermatol Venereol (Stockh). 1989; 69:227–233.
6. Yngveson M., Svensson A., Isacsson A. Prevalence of self-reported hand
dermatoses in upper secondary school pupils. Acta Dermatol Venereol (Stockh).
1998; 78:371–374.
7. Ramsing D.W., Agner T. Effect of glove occlusion on human skin. I: Shortterm

experimental exposure. Contact Dermatitis. 1996; 34:1–5.
8. Ramsing D.W., Agner T. Effect of glove occlusion on human skin. II: Longterm
experimental exposure. Contact Dermatitis. 1996; 34:258–262.
9. Nilsson E. Individual and environmental risk factors for hand eczema in hospital
workers. Acta Dermatol Venereol (Stockh). 1986; suppl 128:1–63.
10. Forrester B.G., Roth V.S. Hand dermatitis in intensive care units. J Occup Environ
Med. 1998; 40:881–885.
11. Malten K.E., den Arend J.A. Irritant contact dermatitis. Traumatic and cumulative
impairment by cosmetics, climate, and other daily loads. Derm Beruf Umwelt.
1985; 33:125–132.
12. Berne B., Bostrom A., Grahnen A.F., Tammela M. Adverse effects of cosmetics
and toiletries reported to the Swedish Medical Products Agency 1989– 1994.
Contact Dermatitis. 1996; 34:359–362.
13. de Groot A.C., Weyland J.W., Nater J.P. Unwanted Effects of Cosmetics and
Drugs Used in Dermatology, Amsterdom: Elsevier, 1994.
14. Willis C.M., Shaw S., De Lacharrière O., Baverel M., Reiche L., Jourdain R.,
Bastien P., Wilkinson J.D. Sensitive skin: an epidemiological study. Br J Dermatol.
2001; 145:258–263.
15. Mathias C.G.T., Maibach H.I. Dermatotoxicology monographs. I. Cutaneous
irritation: factors influencing the response to irritants. Clin Toxicol 1978; 13: 333–
346.
16. Basketter D., Gerberick F., Kimber I., Willis C. Contact irritation mechanisms. In:
D.Anderson, M.D.Waters, T.C.Mars, Toxicology of Contact Dermatitis. Allergy,
Irritancy and Urticaria, John Wiley & Sons, London, 1999; 11–38.
17. Malten K.E. Thoughts on irritant contact dermatitis. Contact Dermatitis 1981; 7:
238–247.
18. Loden M., Lindberg M. Moisturisers and irritant contact dermatitis. In: A.L. Chew,
H.I.Maibach, eds. Handbook of Irritant Dermatitis, SpringerVerlag, Berlin, in press
(2003).
19. Wigger-Alberti W., Elsner P. Contact dermatitis due to irritation. In: Kanerva L.
Elsner P.Wahlberg J.E.Maibach H.I. Handbook of Occupational Dermatology.
Springer-Verlag, Berlin 2000:99–110.
20. Berg M., Lonne-Rahm S.B., Fischer T. Patients with visual display unit-related
facial symptoms are stingers. Acta Dermatol Venereol (Stockh) 1998; 78:44–45.
21. Lonne-Rahm S.B., Fischer T., Berg M. Stinging and rosacea. Acta Dermatol
22. Seidenari S., Francomano M., Mantovani L. Baseline biophysical parameters in
subjects with sensitive skin. Contact Dermatitis 1998; 38: 311–315.
23. Wahlberg J.E. Clinical overview of irritant dermatitis. In: P.G.M. van der Valk,
H.I.Maibach, eds. The Irritant Contact Dermatitis Syndrome, Boca Raton, FL CRC
Press, 1996:1–6.
24. Lammintausta K., Maibach H. Exogenous and endogenous factors in skin
irritation. Int J Dermatol 1988; 27:213–222.
25. Dahl M.V. Chronic irritant contact dermatitis: mechanisms, variables, and
differentiation from other forms of contact dermatitis. Adv Dermatol 1988; 3: 261–
275.
26. Berardesca E., Distante F. The modulation of skin irritation. Contact Dermatitis
1994; 31:281–287.
27. Wilhelm K.P., Maibach H.I. Factors predisposing to cutaneous irritation. Dermatol
Clin 1990; 8:17–22.
28. Björnberg A. Skin reactions to primary irritants in patients with hand eczema.,
Göteborg: Oscar IsacsonTryckeri, 1968.
29. Robinson M.K. Population differences in acute skin irritation responses. Race, sex,
age, sensitive skin and repeat subject comparisons. Contact Dermatitis 2002; 46:86–
93.
30. Basketter D.A., Griffiths H.A.,Wang X.M.,Wilhelm K.P., McFadden J. Individual,
ethnic and seasonal variability in irritant susceptibility of skin: the implications for
a predictive human patch test. Contact Dermatitis 1996; 35: 208–213.
31. Effendy I., Loeffler H., Maibach H.I. Baseline transepidermal water loss in patients
with acute and healed irritant contact dermatitis. Contact Dermatitis 1995; 33:371–
374.
32. Agner T. Basal transepidermal water loss, skin thickness, skin blood flow and skin
colour in relation to sodium lauryl sulphate-induced irritation in normal skin.
33. Tupker R.A., Pinnagoda J., Coenraads P.J., Nater J.P. Susceptibility to irritants:
role of barrier function, skin dryness and history of atopic dermatitis. Br J
Dermatol 1990; 123:199–205.
34. Iliev D., Hinnen U., Elsner P., Lliev D. Clinical atopy score and TEWL are not
correlated in a cohort of metalworkers. Contact Dermatitis 1997; 37: 235–236.
35. Loffler H., Effendy I. Skin susceptibility of atopic individuals. Contact Dermatitis
1999; 40:239–242.
36. Stolz R., Hinnen U., Elsner P. An evaluation of the relationship between ‘atopic
skin’ and skin irritability in metalworker trainees. Contact Dermatitis 1997; 36:281–
284.
37. Coenraads P.J., Diepgen T.L., Smit J. Epidemiology. In: R.J.G.Rycroft, T. Menné,
P.J.Frosch, J.-P.Lepoittevin, eds. Textbook of Contact Dermatitis ed 3, Berlin,
Springer-Verlag, 2001; 187–206.
38. Yamamoto A., Serizawa S., Ito M., Sato Y. Stratum corneum lipid abnormalities in
39. Schafer L., Kragballe K. Abnormalities in epidermal lipid metabolism in patients
with atopic dermatitis. J Invest Dermatol 1991; 96:10–15.
40. Melnik B., Hollmann J., Hofmann U.,Yuh M.S., Plewig G. Lipid composition of
outer stratum corneum and nails in atopic and control subjects. Arch Dermatol Res
1990; 282:549–551.
41. di Nardo A., Sugino K., Wertz P., Ademola J., Maibach H.I. Sodium lauryl
sulphate (SLS) induced irritant contact dermatitis: a correlation study between
ceramides and in vivo parameters of irritation. Contact Dermatitis 1996; 35:86–91.
42. Forslind B. A domain mosaic model of the skin barrier. Acta Dermatol Venereol
(Stockh) 1994; 74:1–6.
43. Norlén L. Skin barrier structure and function: the single gel phase model. J Invest
Dermatol 2001; 117:830–836.
44. Forslind B., Engstrom S., Engblom J., Norlén L. A novel approach to the
understanding of human skin barrier function. J Dermatol Sci 1997; 14:115–125.
45. Norlén L. Skin barrier formation: the membrane folding model. J Invest Dermatol
2001; 117:823–829.
46. Elias P.M. The stratum corneum revisited. J Dermatol 1996; 23:756–758.
47. Elias P.M. Lipids and the epidermal permeability barrier. Arch Dermatol Res 1981;
270:95–117.
48. Feingold K.R., Elias P.M. The environmental interface: regulation of permeability
barrier homeostasis. In: M.Lodén, H.I.Maibach, eds. Dry Skin and Moisturizers.
Chemistry and Function, CRC Press, Boca Raton, FL, 2000; 45–58.
49. Elias P.M., Nau P., Hanley K., Cullander C., Crumrine D., Bench G., Sideras-
Haddad E., Mauro T., Williams M.L., Feingold K.R. Formation of the epidermal
calcium gradient coincides with key milestones of barrier ontogenesis in the
rodent. J Invest Dermatol 1998; 110:399–404.
50. Mao-Qiang M., Mauro T., Bench G., Warren R., Elias P.M., Feingold K.R.
Calcium and potassium inhibit barrier recovery after disruption, independent of the
type of insult in hairless mice. Exp Dermatol 1997; 6:36–40.
51. Lee S.H., Elias P.M., Proksch E., Menon G.K., Mao-Quiang M., Feingold K.R.
Calcium and potassium are important regulators of barrier homeostasis in murine
epidermis. J Clin Invest 1992; 89:530–538.
52. Forslind B. The skin barrier: analysis of physiologically important elements and
trace elements. Acta Dermatol Venereol (Stockh) suppl 2000; 208:46–52.
53. Forslind B., Pallon J. Particle probes and skin physiology. In: M. Lodén, H.I.
Maibach, eds. Dry Skin and Moisturizers. Chemistry and Function, CRC Press,
Boca Raton, FL: 2000; 71–88.
54. Warner R.R. The distribution and function of physiological elements in skin. In:
M. Lodén, H.I. Maibach eds. Dry Skin and Moisturizers. Chemistry and Function,
CRC Press, Boca Raton, FL: 2000; 89–108.
55. Grubauer G., Elias P.M., Feingold K.R. Transepidermal water loss: the signal for
recovery of barrier structure and function. J Lipid Res 1989; 30:323–333.
56. Nickoloff B.J., Naidu Y. Perturbation ofepidermal barrier function correlates with
initiation of cytokine cascade in human skin. J Am Acad Dermatol 1994; 30:535–
546.
57. Barker J.N.W.N., Mitra R.S., Griffiths C.E.M. Keratinocytes as initiators of
inflammation. Lancet 1991; 337:211–214.
58. Nickoloff B.J., Turka L.A. Keratinocytes: key immunocytes of the integument. Am
J Pathol 1993; 143:325–331.
59. Elias P.M., Wood K.C., Feingold K.R. Epidermal pathogenesis of inflammatory
dermatoses. Am J Contact Dermattis 1999; 10:119–126.
60. Nickoloff B.J. Immunological reactions triggered during irritant contact dermatitis.
Am J Contact Dermatitis 1998; 9:107–110.
61. Lisby S., Baadsgaard O. Mechanisms of irritant contact dermatitis. In:
R.J.G.Rycroft, T.Menné, P.J.Frosch, J.-P.Lepoittevin, eds. Textbook of Contact
Dermatitis, ed 3, Springer-Verlag, Berlin 2001; 91–112.
62. Elias P.M., Feingold K.R. Does the tail wag the dog? Role of the barrier in the
pathogenesis of inflammatory dermatoses and therapeutic implications. Arch
Dermatol 2001; 137:1079–1081.
63 . Pallon J., Malmqvist K.G., Werner-Linde Y, Forslind B. Pixel analysis of
pathological skin with special reference to psoriasis and atopic dry skin. Cell Mol
Biol 1996; 42:111–118.
64. Forslind B., Lindberg M., Roomans G.M., Pallon J., Werner-LindeY. Aspects on
the physiology of human skin: studies using particle probe analysis. Microsc Res
Techniques 1997; 38:373–386.
65. Grängsjö A., Ybo A., Roomans G.M., Lindberg M. Irritant-induced keratinocyte
proliferation evaluated with two different methods: immunohisto-chemistry and X-
ray microanalysis. J Submicrosc Cytol Pathol 2000; 32: 11–16.
66. Grängsjö A., Leijon-Kuligowski A.,Torma H., Roomans G.M., Lindberg M.
Different pathways in irritant contact eczema? Early differences in the epidermal
elemental content and expression of cytokines after application of 2 different
irritants. Contact Dermatitis 1996; 35:355–360.
67. Serup J., Serup G.B.C. Handbook of Non-invasive Methods and the Skin, CRC
Press, Boca Raton, FL, 1995.
68. Elsner P., Berardesca E., Maibach H.I. Water and the Stratum Corneum, CRC
Press, Boca Raton, FL, 1994.
69. Berardesca E., Elsner P., Maibach H.I. Cutaneous Blood Flow, CRC Press, Boca
Raton, FL, 1995.
70. Berardesca E., Elsner P., Wilhelm K.P. Bioengineering of the Skin: Methods and
Instrumentation, CRC Press, Boca Raton, FL, 1995.
71. Pinnagoda J., Tupker R.A., Agner T., Serup J. Guidelines for transepidermal water
loss (TEWL) measurement. A report from the Standardization Group of the
European Society of Contact Dermatitis. Contact Dermatitis 1990; 22:164–178.
72. Bircher A., de Boer E.M., Agner T., Wahlberg J.E., Serup J. Guidelines for
measurement of cutaneous blood flow by laser Doppler flowmetry. A report from
the Standardization Group of the European Society of Contact Dermatitis. Contact
Dermatitis 1994; 30:65–72.
73. Fullerton A., Fischer T., Lahti A., Wilhelm K.P., Takiwaki H., Serup J. Guidelines
for measurement of skin colour and erythema. A report from the Standardization
Group of the European Society of Contact Dermatitis. Contact Dermatitis 1996; 35:
1–10.
74. Willis C.M., Stephens C.J.M., Wilkinson J.D. Epidermal damage induced by
irritants in man: a light and electron microscopic study. J Invest Dermatol 1989; 93:
695–699.
75. Parish W.E. Inflammation. In: R.H. Champion, J.L. Burton, D.A. Burns, S.M.
Breathnach, Textbook of Dermatology, ed 6, Blackwell Scientific Publications,
Oxford, 1998; 230.
76. Fartasch M., Schnetz E., Diepgen T.L. Characterization of detergent-induced
barrier alterations—effect of barrier cream on irritation. J Invest Dermatol 1999;
Symp Proc 3(2): 121–127.
77. Nagao S., Stroud J.D., Hamada T., Pinkus H., Birmingham D.J. The effect of
sodium hydroxide and hydrochloric acid on human epidermis. Acta Dermatol
78. Willis C.M., Stephens C.J.M., Wilkinson J.D. Differential patterns of epidermal
leukocyte infiltration in patch test reactions to structurally unrelated chemical
irritants. J Invest Dermatol 1993; 101:364–370.
79. Forslind B. Particle probe analysis in the study of skin physiology. Scan Electron
Microsc 1986; III: 1007–1014.
80. Forslind B., Lindberg M., Malmqvist K.G., Pallon J., Roomans G.M., Werner-
Linde Y. Human skin physiology studied by particle probe microanalysis. Scan
Microsc 1995; 9:1011–1025.
81. Grängsjö A. Epidermal keratinocytes studied by X-ray microanalysis, with special
reference to contact dermatitis. Thesis. Acta Universitatis Upsaliensis 1999; 884:1–
31.
82. Bieber T., Ring J., Braun-Falco O. Comparison of different methods for
enumeration of Langerhans cells in vertical cryosections of human skin. Br J
Dermatol 1988; 118:385–392.
83. Kimber I., Cumberbatch M. Stimulation of Langerhans cell migration by tumor
necrosis factor-alpha (TNF-alpha). J Invest Dermatol 1992; 99: 48S-50S.
84. Lundqvist F.N., Bäck O. Interleukin-1 decreases the number of Ia+ epidermal cells
but increases their expression of Ia antigen. Acta Dermatol Venereol (Stockh) 1990;
70:391–394.
85. Sporn M.B., Roberts A.B. Peptide growth factors are multifunctional. Nature 1988;
332:217–219.
86. Corsini E., Galli C.L. Epidermal cytokines in experimental contact dermatitis.
Toxicology 2000; 142:203–211.
87. Kupper T.S., Grove R.W. The interleukin-1 axis and cutaneous inflammation. J
Invest Dermatol 1995; 105 suppl: 62S-66S.
88. Gordon J.R., Galli S.J. Mast cells as a source of both preformed and immuno-
logically inducible TNF-alpha/cachectin. Nature 1990; 346:274–276.
89. Lisby S., Muller K.M., Jongeneel C.V., Saurat J.-H., Hauser C. Nickel and skin
irritants up-regulate tumor necrosis factor-a mRNA in keratinocytes by different but
potentially synergistic mechanism. Int Immunol 1995; 7: 343–352.
90. Müller-Decker K., Heinzelmann T., Furstenberger G., Kecskes A., Lehmann W.-
D. Arachidonic acid metabolism in primary irritant dermatitis produced by patch
testing of human skin with surfactants. Toxicol Appl Pharmacol 1998; 153:59–67.
91. Kupper T.S. A model for inducible cytokine production by non-bone marrow-
derived cells in cutaneous inflammatory and immune responses. J Invest Dermatol
1990; 94:146S-150S.
92. Friedmann P.S., Strickland I., Memon A.A., Johnson P.M. Early time course of
recruitment of immune surveillance in human skin after chemical provocation. Clin
Exp Immunol 1993; 9:351–356.
93. Rothlein R., Dustin M.L., Marlin S.D., SpringerT.A. A human intercellular
adhesion molecule (ICAM-1) distinct from LFA-1. J Immunol 1986; 137: 1270–
1274.
94. Lange Vejlsgaard G., Ralfkiaer E., Avnstorp C., Czajkowski, Merlin S.D., Rothlein
R. Kinetics and characterization of intercellular adhesion molecule-1 (ICAM-1)
expression on keratinocytes in various inflammatory skin lesions and malignant
lymphomas. J Am Acad Dermatol 1989; 20: 782–790.
95. Lindberg M., Färm G., Scheynius A. Differential effects of sodium lauryl sulphate
and nonanoic acid on the expression of CD1 and ICAM-1 in human epidermis.
Acta Dermatol Venereol (Stockh) 1991; 71:384–388.
96. Willis C.M., Stephens C.J.M., Wilkinson J.D. Selective expression of immune-
associated surface antigens by keratinocytes in irritant contact dermatitis. J Invest
Dermatol 1991; 96:505–511.
97. Flier J., Boorsma D.M., Bruynzeel D.P., van Beek P.J., Stoof T.J., Scheper R.J.,
Willemze R., Tensen C.P. The CXCR3 activating chemokines IP-10, Mig, and IP-9
are expressed in allergic but not in irritant patch test reactions. J Invest Dermatol
1999; 113:574–578.
98. Dustin M.L., Singer K.H., Tuck D.T., Springer T.A. Adhesion of T lymphocytes to
epidermal keratinocytes is regulated by interferon gamma and is mediated by
intercellular adhesion molecule 1 (ICAM-1). J Exp Med 1988; 167:1323–1340.
99. Griffiths C.E.M., Voorhees J.J., Nickoloff B.J. Characterization of inter-cellular
adhesion molecule-1 and HLA-DR expression in normal and inflamed skin:
Modulation by recombinant gamma interferon and tumor necrosis factor. J Am
Acad Dermatol 1989; 20:617–629.
100. Darr D., Fridovich I. Free radicals in cutaneous biology. J Invest Dermatol 1994;
102:671–675.
101. Ikeda M., Schroeder K.K., Mosher L.B.,Woods C.W., Akeson A.L. Suppressive
effect of antioxidants on intercellular adhesion molecule-1 (ICAM-1) expression in
human epidermal keratinocytes. J Invest Dermatol 1994; 103: 791–796.
102. Lange R.W., Hayden P.J., Chignell C.F., Luster M.I. Anthralin stimulates
keratinocyte-derived proinflammatory cytokines via generation of reactive oxygen
species. Inflamm Res 1998; 47:174–181.
103. Andrew P.J., Haram H., Lindley I.J. Up-regulation of interleukin-1 betastimulated
interleukin- 8 in human keratinocytes by nitric oxide. Biochem Pharmacol 1999;
57:1423–1429.
104. Muller K.M., Wiegrebe W., Younes M. Formation of active oxygen species by
dithranol. III. Dithranol, active oxygen species and lipid peroxidation in vivo. Arch
Pharm 1987; 320:59–66.
105. Fuchs J., Packer L. Investigation of anthralin free radicals in model systems and in
skin of hairless mice. J Invest Dermatol 1989; 92:677–682.
106. Willis C.M., Reiche L., Wilkinson J.D. Immunocytochemical demonstration of
reduced Cu,Zn-superoxide dismutase levels following topical application of
dithranol and sodium lauryl sulphate: an indication of the role of oxidative stress in
acute irritant contact dermatitis. Eur J Dermatol 1998; 8:8–12.
107. Willis C.M., Britton L.E., Reiche L., Wilkinson J.D. Reduced levels of glutathione
S-transferases in patch test reactions to dithranol and sodium lauryl sulphate as
demonstrated by quantitative immunocytochemistry: evidence for oxidative stress
in acute irritant contact dermatitis. Eur J Dermatol 2001; 11:99–104.
108. Aioi A., Shimizu T., Kuriyama K. Effect of squalene on superoxide anion
generation induced by a skin irritant, lauroylsarcosine. Int J Pharm 1995; 113:159–
164.
109. Fuchs J., Milbradt R. Antioxidant inhibition of skin inflammation induced by
reactive oxdants: evaluation of the redox couple dihydrolipoate/lipoate. Skin
Pharmacol 1994; 7:278–284.
110. van Duijnhoven-Avontuur W.M.G., Alkemade J.A.C., Schalkwijk J., Mier P.D.,
van der Valk P.G.M. The inflammatory and proliferative response of normal skin in
a model for acute chemical injury: ornithine decarboxylase induction as a common
feature in various models of acute skin injury. Br J Dermatol 1994; 130:725–730.
111. De Zwart A.J., de Jong E.M.G.J., van de Kerkhof P.C.M. Topical application of
dithranol on normal skin induces epidermal hyperproliferation and increased Ks8.
12 binding. Skin Pharmacol 1992; 5:34–40.
112. Le T.K.M., van der Valk P.G.M., Schalkwijk J., van der Kerkhof P.C.M. Changes
in epidermal proliferation and differentiation in allergic and irritant contact
dermatitis reactions. Br J Dermatol 1995; 133:236–240.
113. Willis C.M., Stephens C.J.M., Wilkinson J.D. Differential effects of structurally
unrelated chemical irritants on the density of proliferating keratinocytes in 48 h
patch test reactions. J Invest Dermatol 1992; 99:449–453.
114. Wilhelm K.-P., Saunders J.C., Maibach H.I. Increased stratum corneum turnover
induced by subclinical irritant dermatitis. Br J Dermatol 1990; 122: 793–798.
115. LeT.K.M., Schalkwijk J., Siegenthaler G., van der Kerkhof P.C.M., Veerkamp
J.H., van der Valk P.G.M. Changes in keratinocyte differentiation following mild
irritation by sodium dodecyl sulphate. Arch Dermatol Res 1996; 288: 684–690.
116. De Mare S., van Erp P.E.J., Ramaekers F.C.S., van de Kerkhof P.C.M. Flow
cytometric quantification of human epidermal cells expressing keratin 16 in vivo
after standardized trauma. Arch Dermatol Res 1990; 282: 126–130.
117. Tupker R.A., Pinnagoda J., Coenraads P.J., Nater J.P. The influence of repeated
exposure to surfactants on the human skin as determined by transepidermal water
loss and visual scoring. Contact Dermatitis 1989; 20: 108–114.
118. Frosch P.J., Kurte A., Pilz B. Efficacy of skin barrier creams. III. The repetitive
irritation test (RIT) in humans. Contact Dermatitis 1993; 29:113–118.
119. Frosch P.J., Kurte A. Efficacy of skin barrier creams. IV. The repetitive irritation
test (RIT) with a set of 4 standard irritants. Contact Dermatitis 1994; 31:161–168.
120. Schnetz E., Diepgen T.L., Elsner P., Frosch P.J., Klotz A.J., Kresken J., Kuss O.,
Merk H., Schwanitz H.J., Wigger-Alberti W., Fartasch M. Multicentre study for the
development of an in vivo model to evaluate the influence of topical formulations
on irritation. Contact Dermatitis 2000; 42:336–343.
121. Wigger-Alberti W., Caduff L., Burg G., Elsner P. Experimentally induced chronic
irritant contact dermatitis to evaluate the efficacy of protective creams in vivo. J
Am Acad Dermatol 1999; 40:590–596.
122. Ale S.I., Laugier J.P., Maibach H.I. Differential irritant skin responses to tandem
application of topical retinoic acid and sodium lauryl sulphate. II. Effect of time
between first and second exposure. Br J Dermatol 1997; 137: 226–233.
123. Effendy I., Weltfriend S., Patil S., Maibach H.I. Differential irritant skin responses
to topical retinoic acid and sodium lauryl sulphate alone and in crossover design.
Br J Dermatol 1996; 134:424–430.
124. Kappes U.P., Göritz N.,Wigger-Alberti W., Heinemann C., Elsner P. Tandem
application of sodium lauryl sulphate and n-propanol does not lead to enhancement
of cumulative skin irritation. Acta Dermatol Venereol (Stockh) 2001; 81:403–405.
125. Wigger-Alberti W., Krebs A., Elsner P. Experimental irritant contact dermatitis due
to cumulative epicutaneous exposure to sodiutn lauryl sulphate and toluene: single
and concurrent application. Br J Dermatol 2000; 143: 551–556.
126. Clarys P., Manou I., Barel A.O. Influence of temperature on irritation in the hand/
forearm immersion test. Contact Dermatitis 1997; 36:240–243.
127. Berardesca E., Vignoli G.P., Distante E, Brizzi P, Rabbiosi G. Effects of water
temperature on surfactant-induced skin irritation. Contact Dermatitis 1995; 32:83–
87.
128. Ohlenschlaeger J., Friberg J., Ramsing D., Agner T. Temperature dependency of skin
susceptibility to water and detergents. Acta Dermatol Venereol (Stockh) 1996; 76:
274–276.
129. Löffler H., Aramaki J., Effendy I. Response to thermal stimuli in skin pretreated
with sodium lauryl sulphate. Acta Dermatol Venereol (Stockh) 2001; 81:395–397.
130. Schaefer H., Redelmeier T.E. Skin Barrier, Principles of Percutaneous Absorption,
Basel, Karger, 1996.
131. Zhai H., Maibach H.I. Skin occlusion and irritant and allergic contact dermatitis: an
overview. Contact Dermatitis 2001; 44:201–206.
132. Van der Valk P.G., Maibach H.I. Post-application occlusion substantially increases
the irritant response of the skin to repeated short-term sodium lauryl sulphate (SLS)
exposure. Contact Dermatitis 1989; 21:335–338.
133. Matsumura H., Oka K., Umekage K., Akita H., Kawai J., Kitazawa Y, Suda
S.,Tsubota K., Ninomiya Y., Hirai H. Effect of occlusion on human skin. Contact
Dermatitis 1995; 33:231–235.
134. Ghadially R. Aging and the epidermal permeability barrier: implications for
contact dermatitis. Am J Contact Dermatitis 1998; 9:162–169.
135. Schwindt D.A., Wilhelm K.P., Miller D.L., Maibach H.I. Cumulative irritation in
older and younger skin: a comparison. Acta Dermatol Venereol (Stockh) 1998; 78:
279–283.
136. Lammintausta K., Maibach H.I., Wilson D. Irritant reactivity in males and females.
137. Patil S., Maibach H.I. Effect of age and sex on the elicitation of irritant contact
dermatitis. Contact Dermatitis 1994; 30:257–264.
138. Dupuis D., Rougier A., Lotte C.,Wilson D.R., Maibach H.I. In vivo relationship
between percutaneous absorption and transepidermal water loss according to
anatomic site in man. J Soc Cosmet Chem 1986; 37:351–357.
139. Elias P.M., Cooper E.R., Korc A., Brown B.E. Percutaneous transport in relation to
stratum corneum structure and lipid composition. J Invest Dermatol 1981; 76:297–
301.
140. Van der Valk P.G., Maibach H.I. Potential for irritation increases from the wrist to
the cubital fossa. Br J Dermatol 1989; 121:709–712.
141. Yosipovitch G., Xiong G.L., Haus E., Sackett-Lundeen L., Ashkenazi I., Maibach
H.I. Time-dependent variations of the skin barrier function in humans:
transepidermal water loss, stratum corneum hydration, skin surface pH, and skin
temperature. J Invest Dermatol 1998; 110:20–23.
142. Freeman S., Maibach H.I. Study of irritant contact dermatitis produced by repeat
patch test with sodium lauryl sulfate and assessed by visual methods,
transepidermal water loss, and laser Doppler velocimetry. J Am Acad Dermatol
1988; 19:496–502.
143. Werner Y., Lindberg M. Transepidermal water loss in dry and clinically normal
skin in patients with atopic dermatitis. Acta Dermatol Venereol (Stockh) 1985; 65:
102–105.
144. van der Valk G.M., Nater J.P., Bleumink E. Vulnerability of the skin to surfactants
in different groups of eczema patients and controls as measured by water vapour
loss. Clin Exp Dermatol 1985; 10:98–103.
145. Agner T. Susceptibility of atopic dermatitis patients to irritant dermatitis caused by
sodium lauryl sulphate. Acta Dermatol Venereol (Stockh) 1991; 71: 296–300.
146. Nassif A., Chan S.C., Storres F.J., Hanifin J.M. Abnormal skin irritancy in atopic
dermatitis and in atopy without dermatitis. Arch Dermatol 1994; 130: 1402–1407.
147. Cowley N.C., Farr P.M. A dose-response study of irritant reactions to sodium lauryl
sulphate in patients with seborrhoeic dermatitis and atopic eczema. Acta Dermatol
148. Al Jaberi H., Marks R. Studies of the clinically uninvolved skin in patients with
dermatitis. Br J Dermatol 1984; 111:437–443.
149. Berndt U., Hinnen U., Iliev D., Elsner P. Hand eczema in metalworker trainees—an
analysis of risk factors. Contact Dermatitis 2000; 43:327–332.
150. Grimnes S., Martinse Ö.G. Skin and keratinised tissue In: Bioimpedance and
Bioelectricity Basics, Academic Press, San Diego, CA, 2000, 294– 300.
151. Mahmoud G., Lachapelle J.-M., Van Neste D. Histological assessment of skin
damage by irritants: its possible use in the evaluation of a “barrier-cream”. Contact
Dermatitis 1984; 11:179–185.
152. Metz J. Elecktronenmikroskopische Untersuchungen an allergischen und toxischen
Epicutantestreaktionen des Menschen. Arch Derm Forsch 1972; 245:125–146.
153. Fischer J.P., Cooke R.A. Experimental toxic and allergic contact dermatitis. J
Allergy 1958; 29:411–428.
154. Nater J.P., Hoedemaeker Ph.J. Histological differences between irritant and allergic
patch test reactions in man. Contact Dermatitis 1976; 2:247–253.
155. Nater J.P., Baar A.J.M., Hoedemaeker Ph.J. Histological aspects of skin reactions
to propylene glycol. Contact Dermatitis 2002; 3:181–185.
156. Johannesson A., Hammar H. Corneocyte morphology and formation rate in lichen
planus and experimental parakeratosis in subjects with and without psoriasis. Acta
Dermatol Venereol (Stockh) 1982; 62:463–470.
157. Lindberg M., Johannesson A., Forslind B. The effect of occlusive treatment on
human skin: an electron microscopic study on epidermal morphology as affected by
occlusion and dansyl chloride. Acta Dermatol Venereol (Stockh) 1982; 62:1–5.
158. Lupulescu A.P., Birmingham D.J., Pinkus H. An electron microscopic study of
human epidermis after acetone and kerosene administration. J Invest Dermatol
1973; 60:33–45.
159. Mahmoud G., Lachapelle J.-M. Evaluation expérmentale de l’efficacité de crêmes
barrière et de gels antisolvants dans laprévention de l’irritation cutanée provoquée
par des solvantsorganiques. Cah Med Trav 1985; 22:163–168.
160. Willis C.M., Young E., Brandon D.R., Wilkinson J.D. Immunopathological and
ultrastructural findings in human allergic and irritant contact dermatitis. Br J
Dermatol 1986; 115:305–316.
161. Kanerva L., Ranki A., Mustakallio K., Lauharanta J. Langerhans cell-mononuclear
cell contacts are not specific for allergy in patch tests. Br J Dermatol (suppl 25)
1983; 64–67.
162. Kolde G., Knop J. Different cellular reaction patterns of epidermal Langerhans
cells after application of sensitizing, toxic, and tolerogenic compounds. A
comparative ultrastructural and morphometric time-course analysis. J Invest
Dermatol 1987; 89:19–23.
163. Gerberick G.F., Rheins L.A., Ryan C.A., Ridder G.M., Haren M., Miller C.,
Oelrich D.M., von Bargen E. Increases in human epidermal DR+CD1+, DR+CD1-
CD36+, and DR-CD3+cells in allergic versus irritant patch test responses. J Invest
Dermatol 1994; 103:524–529.
164. Falck B., Andersson A., Elofsson R., Sjöborg S. New views on epidermis and its
Langerhans cells in the normal state and in contact dermatitis. Acta Dermatol
Venereol (Stockh) 1981; suppl 99:3–27.
165. Ferguson J., Gibbs J.H., Swanson Beck J. Lymphocyte subsets and Langerhans
cells in allergic and irritant patch test reactions: histometric studies. Contact
Dermatitis 1985; 13:166–174.
166. Marks J.G., Zaino R.J., Bressler M.F., Williams J.V. Changes in lymphocyte and
Langerhans cell populations in allergic and irritant contact dermatitis. Int J
Dermatol 1987; 26:354–357.
167. Lisby S., Baardsgaard O., Cooper K.D., Lange Vejlsgaard G. Decreased number
and function of antigen-presenting cells in the skin following application of irritant
agents: relevance for skin cancer? J Invest Dermatol 1989; 92: 842–847.
168. Gawkrodger D.J., McVittie E., Carr M.M., Ross J.A., Hunter J.A.A. Phenotypic
characterization of the early cellular response in allergic and irritant contact
dermatitis. Clin Exp Immunol 1986; 66:590–598.
169. Willis C.M., Stephens C.J.M., Wilkinson J.D. Differential effects of structurally
unrelated chemical irritants on the density and morphology of epidermal CD1
+cells. J Invest Dermatol 1990; 95:711–716.
170. Lindberg M., Emtestam L. Dynamic changes in the epidermal OKT6 positive cells
at mild irritant reactions in human skin. Acta Dermatol Venereol (Stockh) 1986; 66:
117–120.
171. Avnstorp C., Ralfkiaer E., Jörgensen J., Lange Wantzin G. Sequential
immunophenotypic study of lymphoid infiltrate in allergic and irritant reactions.
172. Kanerva L., Ranki A., Lauharanta J. Lymphocytes and Langerhans cells in patch
tests: an immunohisfochemical and electron microscopic study. Contact Dermatitis
1984; 11:150–155.
173. Scheynius A., Fischer T., Forsum U., Klareskog L. Phenotypic characterization in
situ of inflammatory cells in allergic and irritant contact dermatitis. Clin Exp
Dermatol 1984; 55:81–92.
174. Christensen O.B., Wall L.M. Long term effect on epidermal dendritic cells of four
different types of exogenous inflammation. Acta Dermatol Venereol (Stockh)
1987; 67:305–309.
175. Ulfgren A.K., Klareskog L., Lindberg M. An immunohistochemical analysis of
cytokine expression in allergic and irritant contact dermatitis. Acta Dermatol
176. Oxholm A., Oxholm P., Avnstorp C., Bendtzen K. Keratinocyte expression of
interleukin-6 but not of tumour necrosis factor-alpha is increased in the allergic and
the irritant patch test reaction. Acta Dermatol Venereol (Stockh) 1991; 71:93–98.
177. Morhenn V.B., Chang E.Y., Rheins L.A. A non-invasive method for quantifying
and distinguishing inflammatory skin reactions. J Am Acad Dermatol 1999; 41:
687–692.
178. Hunziker T., Brand C.U., Kapp A., Waelti E.R., Braathen L.R. Increased levels of
inflammatory cytokines in human skin lymph from sodium lauryl sulphate-induced
contact dermatitis. Br J Dermatol 1992; 127:254–257.
179. Müller-Decker K., Heinzelmann T., Furstenberger G., Kecskes A., Lehmann W.-
D. Arachidonic acid metabolism in primary irritant dermatitis produced by patch
testing of human skin with surfactants. Toxicol Appl Pharmacol 1998; 153:59–67.
180. Kassis V., Mortensen T., Söndergaard J. Prostaglandin E1 in suction-separated

human epidermal tissue in primary irritant dermatitis. Acta Dermatol Venereol
(Stockh) 1981; 61:429–431.
181. Kobza Black A., Barr R.M., Wong W., Brain S., Greaves M.W., Dickinson R.,
Shroot B., Hensby C.N. Lipoxygenase products of arachidonic acid in human
inflamed skin. Br J Clin Pharmacol 1985; 20:185–190.
182. Søndergaard J., Greaves M.W., Jørgensen H.P. Recovery of prostaglandins in
human primary irritant dermatitis. Arch Dermatol 1974; 110:556–558.
11
Formation and Structure: An Introduction to
Hair
Magnus Lindburg
Stockholm, Sweden
Bo Forslind†
The hair follicle is the structural unit responsible for the formation and
production of a hair fiber. Hair follicles are infoldings of the superficial
epithelium enclosing a dermal part, the dermal papilla. The production of hair is
an important function, and hair has been the foundation for the survival and
evolution of many mammalian species. In man, however, hairs has no vital
function but plays important roles in social and sexual communications. This is
reflected in the interest in hair cosmetics and also in the number of persons
seeking medical help and advise regarding hair problems.
From an evolutionary point of view it is clear that the type of hair produced by
individual follicles at different times of the year or in different phases of sexual
maturation must change [1, 2]. For this reason, all hair follicles are not under
identical control mechanisms. The hair follicles show intermittent activity, with a
cyclic growth pattern in the form of alternating active and resting phases. Each
hair grows to a maximum length, whereupon growth stops; then the hair is
retained for a while before it is shed and eventually replaced. The growth rhythm
of the follicle is modified by circulating hormones such as androgens and thyroid
hormones. Under normal conditions in humans, the number of hair follicles is
established in fetal life, and as the body size increases, the density of follicles
decreases [3, 4]. The total number of hair follicles adds up to about 5 million at
birth. There is a considerable variation in number of follicles with body location,
but the majority of follicles are on the head. With advancing age there is an
increasing loss of hair follicles on the scalp [5].
Structure—function relations of the hair follicle can be viewed in different
ways-from a macroscopic perspective through cellular and molecular aspects
down to genetic considerations. These different perspectives need different
investigative approaches, and a multitude of techniques, such as light
microscopy, transmission (TEM) and scanning electron (SEM) microscopy,
other biophysical techniques, biochemistry, immune techniques, cell cultures of
†Deceased.
252 LINDBERG AND FORSLIND
follicle cells, molecular engineering, and gene techniques, have been applied to
explore the structure and function of the hair follicle and the hair fiber. This chapter
does not attempt to cover all these aspects; rather, it comprises a short
introduction to the structure-function relationships of different parts of the hair
follicle. Subsequent chapters cover hair color, surface structure, growth cycle,
growth control, effects of androgens, and hair loss (alopecia) in more detail. For
further reading on hair, References (1, 2, and 6 to 19) are suggested.
1
HAIR FOLLICLES AND HAIR GROWTH
Hair growth is a cyclic event with stages of growth, growth arrest, and shedding,
followed by the formation of a new hair. In anagen (the growth phase), the hair
fiber is produced from the hair bulb. In the lower part of the follicle, at the level
of the midpart of the hair bulb, matrix cells are located just above the dermal
papilla. This germinative cell population is very active, and following mitosis the
cells differentiate to form the different compartments of the hair shaft namely:
the medulla, cortex, cuticle and the inner root sheath (Fig. 1). In catagen (growth
arrest) the matrix cells undergo apoptosis (programmed cell death), and there is
regression of the hair bulb [18]. The term “keratinization” has been used to
describe the final differentiation of all cellular components of the hair follicle,
although they differ both in morphology and in their biochemical constituents. It
has been suggested that the process be called “consolidation”, instead, to avoid
confusion [20].
The growth rate of human scalp fibers is approximately 0.4 mm/per day [21].
The complete process of consolidation (keratinization) comprises cell division,
protein synthesis and differentiation, and catabolic breakdown of nucleic acids,
cell organelles, and so on. This means that the differentiation of the cell into a
completely keratinized cortex, or cortical cell, occurs within 1 mm from the
matrix cells in the hair bulb. Thus, it takes approximately 2.5 days for a cortex
cell to pass from mitosis to the final state of a cell completely filled with fibrous
and amorphous protein and partially deprived of water. This speed of the
differentiation phase with a high metabolic activity explains why the hair is
sensitive to metabolic interventions such as the administration of cytostatic drugs
used in cancer therapy.
The gross structure of the follicle also determines the shape of the hair
produced. A straight follicle results in a straight hair and a structurally curved
follicle produces a curly hair [22]. Follicle shape also affects the cross-sectional
form of the hair fiber, which is reflected in round or oval body hairs and the
more contorted cross-sectional shape of axillar and pubic hair, which increases
the surface area exposed to apocrine secretions [1].
The root sheath is composed of an inner root sheath (IRS), which is already
present and consolidated (keratinized) as far down in the follicle as the midbulb,
and an outer root sheath (ORS). The origin of the ORS has not been completely
HAIR STRUCTURE AND FORMATION 253
FIGURE 1 Schematic drawing of the dermal part of the hair follicle. The collagen fibers
outside the outer root sheath prevent sideways expansion of the follicle. (Adapted from an
original drawing by Bo Forslind).
determined. In the upper part of the follicle the ORS is continuous with the
interfollicular epidermis. Above the hair bulb the ORS is reinforced by
connective tissue structures (collagen fibers), preventing sideways expansion of
the follicle. The construct of a rigid root sheath is a necessity for the production
of a normal hair shaft.
The domelike shape of the papilla may fulfill two functional purposes: (1) to
provide an augmented matrix cell-dermal interface, which facilitates the presence
of a high number of germinative cells needed for the fast growing hair fiber, and
(2) to give mechanical support, forcing the dividing cells to move outward along
the axis of the growing hair fiber. Pigment-producing melanocytes are also found
in the matrix region. In human hair, pigment is found in medulla and cortex cells
but not in cuticle cells.
In the matrix region the initially soft, nonkeratinized cells are forced to take on
a longitudinal shape when they are pressed upward in the funnellike root sheath
FIGURE 2 Schematic presentation of the funnel part of the hair follicle, indicating the
alignment of the cortex and cuticle cells in the growth direction of the fiber.
(Fig. 2). At this level the keratin intermediate fibers are oriented along the
longitudinal axis of the cell and thus of the hair fiber [1, 20].
2
HAIR KERATIN
In early pioneering work on protein structure, a number of proteins were
characterized on the basis of their X-ray diffraction patterns [23, 24]. Two major
classes of proteins were distinguished, the KMEF (keratin, myosin, epidermin,
fibrin) family and the collagen family. The two major forms of keratins are the -
keratins, which essentially are the keratins of the mammalians, and the -
keratins, which basically are the keratins of the reptiles, and thus of birds. The
keratins of the hair fiber have been separated into three major biochemical
classes: the high sulfur, low molecular weight class, the low sulfur, high
molecular weight class, and the high tyrosine class. The low sulfur, high
molecular weight fraction (45–58 kDa) is composed of proteins in a-helical
configuration; that is, the -helix is the basic structural unit of hair keratin. The
high sulfur, low molecular weight fraction (10–20 kDa) contains proteins present
in a random coil structure. Hence, they are regarded as matrix protein or an
embedding material for the fibrillar, high molecular weight protein fraction. In
human hair the total sulfur content is close to 5% [8], and the hallmark of
keratins is that high sulfur content in both the high and the low sulfur fractions.
The fibrillar keratins belong to a family of structural proteins called
intermediate filaments (IFs), which contains several classes, including, type I
(acidic keratins) and type II (basic keratins). The keratin intermediate filaments
(KIFs) of types I and II are the major products of epidermis and its derivatives
[25, 26]; KIF can be further be divided into subgroups.
The structural protein of the hair follicle is present in -helical arrangement
[27] embedded in a nonhelical matrix composed of proteins called IF-associated
proteins (IFAPs), which are cysteine-rich (high sulfur) proteins in the human hair
fiber.
The complexity of keratin biochemistry has been demonstrated in several
studies [25]. In addition, there are the complex associations between KIF and
keratin intermediate filament—associatedproteins (KIFAPs), which are thought
to serve as KIF matrix proteins by binding the KIF in tight arrays.
3
THE COMPONENTS OF THE HAIR SHAFT
3.1
The Root Sheath
The root sheaths (ORS and IRS) serve as a mold for the growing hair fiber and
ORS and IRS are the first structures to be consolidated in the hair follicle.
The IRS is composed of three layers. Both the outermost Henley layer and the
middle Huxley layer contain straight filaments (diameter ~ 8 nm) aligned along
the axis of the follicle. This arrangement is compatible with a structure that will
not yield (in the fiber axis direction) on stress. These fila-ments are surrounded
by amorphous protein material first visualized as trichohyalin granules. Directly
on the inner part of Huxley layer is the third layer of the IRS, the IRS cuticle,
apposed to the hair fiber cuticle [28], (Fig. 3). From a functional point of view,
the consolidated IRS cells thus ful-fill the requirement for forming a nonyielding
structure that will impose an elongated form onto the cortex and cuticle cells
when they are pressed through the “funnel” formed by the root sheaths [1, 20].
To ensure elastic properties for the hair fiber the root sheath cells are cast off at
the level of the sebaceous gland.
The ORS is composed of two cell layers of different cell types: a more
peripheral “true” ORS cell and the so-called companion cell cells, which seems
to be more closely associated with the Henley cells of the IRS [28]. Studies with
anti-hair keratin monoclonal antibodies indicate that keratin expression in inner
layer of the ORS differs from that of the medulla, cortex, cuticle, and IRS [29, 30].
3.2
The Cuticle
Mature cuticle cells are thin scales arranged in an overlapping fashion with 5 to
10 cells, each 350 to 450 nm thick [2] to form the cuticle. In a 30 nm gap
between the cell boundaries lie the intercellular lamellae. The cuticle cells can be
FIGURE 3 Schematic presentation of a part of a vertical section through the hair shaft
from the outer root sheath (to the left) to the cortex. The drawing demonstrates the
interdigitating arrangement of the cuticle cells and the cuticle part of the inner root sheath
(IRS).
seen as scales overlapping each other with the free border toward the tip of the
hair fiber (Fig. 4 and 5). When the hair fiber emerges above the skin surface, the
cuticle cells adhere closely to the cortex, exposing a smooth distal free border.
Further out they become ragged, and parts of the front edges are progressively
chipped offs leaving a jagged appearance. The cuticle cells lack elasticity and are
fragile [1, 31] and cannot be stretched like cortex cells. The innermost part of the
IRS and the outermost part of the hair fiber (the cuticle cells) interdigitate and
serve as an anchoring for the growing hair fiber.
The morphology of the cuticle cells does not reveal any fibrous components
under the TEM. Inside the bounding surface membrane (the outer surface)
resides the dense so-called A layer, which has a constant width and to contains
high amounts of sulfur [31]. This layer protects the cuticle from negative effects
of physical and chemical environmental factors. The next two layers, the
exocuticle and the endocuticle, vary in width, and it is the endocuticle that first
suffers the effects of chlorinated water and fungal invasion [9]. Access to this
part of the cell is at the chipped border of the free cuticle edge occurring above
the skin surface as an effect of what is called “weathering.” The cuticle
membrane contains large amount of -( -gluta-myl) -lysine isopeptide cross-
links, which provide good resistance to chemical treatments [32, 33] and have
chemical properties different from those of the cortex membrane complex. This
is because the cuticle membrane contains ornithine and citrulline, not found in
cortex membranes.
FIGURE 4 Schematic presentation of cortex (left) and cuticle (right) cells. The cells are
approximately equal in length (~ 120 µm). The cortex cell is about 25 µm wide, but the
cuticle cell ranges in width from 20 to 80 µm.
The cuticle of the hair fiber provides mechanical protection for the cortex cells
but is also developed to control the water content of the fiber [1, 34–37]. During
the formation of the hair fiber cuticle, including the development of the
exocuticle, several transformation steps occur. The result is that the hair fiber
will expose an outer surface consisting of paired lipid bilayers with an
underlying proteinaceous band, the exocuticle. Furthermore, there will be a
formation between the cuticle cells of a single lamella with a lightly stained
central part that may both provide barrier properties and add to cellular adhesion.
Of the bound lipids in the cuticle 90 % are bound by thioester linkage to the cell
membrane complex [36, 37]. The dominant bound fatty acid is 18-
methyleicosanoic acid, which is a branched fatty acid.
3.3
The Cortex
The size and organization of keratin filaments were demonstrated by
transmission electron microscopy more than four decades ago [38, 39]. The
FIGURE 5 The overlapping arrangement of cuticle cells. The free edge (the distal part)
of the cells becomes more and more affected by loss of material as the hair fiber moves
outward from the skin surface.
physical, tensile strength of hair fibers was later shown to be related to the
filament organization and to the cellular architecture of the cortex cells [40, 41].
The keratin of hair belongs to the -keratins, forming filaments of the
intermediate filament (IF) type (diameter ~ 8 nm). In cortex cells these IFs are
subsequently arranged in fibrillar filament bundles with a diameter of
approximately 300 nm. In a cross-sectional view of hair, the cortex cells can be
shown to contain an abundant mass of fibrils, mostly organized in a coiled
manner as denoted by the “fingerprint whorl pattern,” which corresponds to a
“fishbone” -like pattern in longitudinal ultrathin sections [41]. The functional
implication of such an arrangement is a gain in tensile strength by means of
favorable load distribution. This molecular organization of the cortex keratin
thus allows the cells to be extensively stretched [1, 20, 41].
3.4
The Medulla
Above the hair bulb the medulla cells starts to contain intracellular vacuoles. In
human scalp hairs, sometimes called terminal hairs, the medulla is often an
inconspicuous part of the cross section and can be absent, interrupted, or
fragmented. In hairs such as eyelashes, eyebrows, and gray hair fibers with a
larger diameter, the medulla may be unbroken and continuous along the length
of the fiber.
When the cells of the medulla are present, they are often vacuolated and
contain scattered bundles of randomly dispersed fibrillar material [42]. The
periphery of the vacuoles is coated with an amorphous protein material that
appears as granules in the initial stages of differentiation. This protein contains
almost no cysteine but contains -( -glutamyl) -lysine cross-links, which account

for the very low solubility of the consolidated medulla [27].
4
CONCLUSION
An understanding of structure-function relations is essential for an understanding
of normal and pathological changes in the hair follicle and hair fiber. These
structure-function interactions have been described at different levels of
resolution, from the molecular level to the morphology seen in the light
microscope and by visual inspection. In hair research there has been an
expanding focus from these structure-function perspectives to a view in which
the hair follicle and its function can act as a model for studies on several
biological mechanisms and cellular events such as growth control, differentiation,
cell signaling, programmed cell death (apoptosis), and gene expression.
Moreover, new immunological methods have been developed that enable the use
of monoclonal antibodies to reveal facts about, for example, keratin
differentiation, and an to participate in the expansion of molecular engineering
and gene techniques [11, 12, 18, 19, 43, 44]. This also implies new possibilities
for the study of hair diseases or disorders. Some of these aspects are covered in
succeeding chapters of this book.
ACKNOWLEDGMENT
The manuscript was compiled in accordance with a draft by the late Professor Bo
Forslind. Figures 1, 2, and 3 were produced by Mr. Bernt Eriksson and Figure 4
by Mr. Kalle Forss.
REFERENCES
1. Forslind B. Structure and function of the hair follicle. In: Camacho FM, Randall
VA, Price VH, eds. Hair and Its Disorders. Biology, pathology and management.
London: Martin Dunitz, 2000: Chap 1.
2. Dawber RPR, de Berker D, Wojnarowska F. Disorders of hair. Anatomy and
physiology. In: Champion RH, Burton JL, Burns DA, Breatnach SM, eds. Rook/
Wilkinson/Ebling Textbook of Dermatology. London: Blackwell Science, 1998:
2869–2890.
3. Szabo G. The regional frequency and distribution of hair follicles in human skin. In:
Montagna W, Ellis RA, eds. The Biology of Hair Growth. New York: Academic
Press, 1958:33–38.
4. Szabo G. The regional anatomy of the human integument with special reference to
the distribution of hair follicles, sweat glands and melanocytes. Philos Trans R Soc
Lond Biol 1967; 252:447–485.
5. Giacometti L. The anatomy of the human scalp. In: Montagna W, ed. Advances in
the Biology of Skin, Vol. VI. Ageing. Oxford: Pergamon Press, 1965: 97–120.
6. Montagna W. Structure and Function of Skin. 2nd ed. New York: Academic Press,
1962.
7. Forslind B, Kischer CW, Roomans GM. The Integument. AMF. O’Hare, IL:
Scanning Microscopy, 1985.
8. Forslind B, Li HK, Malmqvist KG, Wiegleb D. Elemental content of anagen hairs
in a normal Caucasian population studies with proton induced X-ray emission
(PIXE). Scan Electron Microsc 1986; I:237–241.
9. Forslind B. Clinical applications of scanning electron microscopy and energy
dispersive X-ray analysis in dermatology—an update. Scan Microsc 1988; 2: 959–
976.
10. Forslind B, Malmqvist KG, Wiren K. Genetic diseases, hair structure and
elemental content. In: Rogers GE et al. eds. The Biology of Wool and Hair. London
and New York: Chapman & Hall, 1988:275–285.
11. de Berker D, Sinclair RD. The hair shaft: normality, abnormality, and genetics.
Clin Dermatol 2001; 19:129–134.
12. Millar SE. Molecular mechanisms regulating hair follicle development. J Invest
Dermatol 2002; 118:216–225.
13. Wuepper KD, Norris DA, Messenger A. The fundamentals of hair biology.
Cutaneous Biology Foundation 41st Symposium on the Biology of Skin,
Snowmass, July 25–29, 1992; J Invest Dermatol 1993; 101:1S–152S.
14. Zelickson AS. Ultrastructure of Normal and Abnormal Skin. Philadelphia: Lea &
Febiger, 1967.
15. Olsen EA. Disorders of Hair Growth. New York: McGraw-Hill, 1994.
16. Camacho FM, Montagna W. Trichology. Diseases of the Pilosebaceous Follicle.
Madrid: Libros Princeps/Aula Media, 1997.
17. Camacho FM, Randall VA, Price VH, eds. Hair and Its Disorders. Biology,
Pathology and Management. London: Martin Dunitz, 2000.
18. Stenn, KS, Paus R. Controls of hair follicle cycling. Physiol Rev. 2001; 81: 449–
494.
19. Paus R, Cotsarelis G. The biology of hair follicles. N Engl J Med 1999; 341: 491–
497.
20. Forslind B. Hair keratin and keratinisation—biochemical and biophysical aspects.
In: Camacho FM, Montagna W, eds. Trichology. Diseases of the Pilosebaceous
Follicle. Madrid: Libros Princeps/Aula Media, 1997: Chap 2.
21. Braun-Falco O. Dynamik des normalen und pathologischen Haarwachtums. Arch
Klin Exp Dermatol 1966; 227:419–452.
22. Lindelöf B, Forslind B, Hedblad M-A, Kaveus U. Human hair form. Morphology
revealed by light and scanning electron microscopy and computer aided three-
dimensional reconstruction. Arch Dermatol 1988; 124:1359–1363.
23. Astbury WT, Woods HJ. X-ray studies of the structure of hair, wool, and related
fibers. II. The molecular structure and elastic properties of hair keratin. Philos
Trans R Soc Lond A 1933:333–394.
24. Astbury WT. On the structure of biological fibers and on the problem of muscle.
The Croonian Lecture 1945. Proc R Soc Lond B 1947; 134:303–328.
25. Steinert PM. Structure, function, and dynamics of keratin intermediate filaments. J
26. Fraser B, McRae T, DAD Parry, Suzuki E. Intermediate filaments in a-keratins.
Proc Natl Acad Sci USA 1985; 83:1179–1183.
27. Fraser B, McRae T. Structure of the a-keratin microfibril. J Mol Biol 1976; 108:
435–452.
28. Orwin DF. The cytology and cytochemistry of the wool follicle. Int Rev Cytol
1979; 60:331–374.
29. Ito M, Tazawa T, Ito K et al. Immunological characteristics and histological
distribution of human hair fibrous proteins: studied with anti-hair monoclonal
antibodies, HKN-2, HKN-4 and HKN-6. J Histochem Cytochem 1986; 34: 269–
275.
30. Ito M, Tazawa T, Shimaza N et al. Cell differentiation in human anagen hair
follicles studied with anti-hair keratin monoclonal antibodies. J Invest Dermatol
1986; 86:563–569.
31. Swift JA. The hair surface. In: Orfanos CE, Montagna W, Stuttgen G, eds. Hair
Research. Berlin: Springer-Verlag, 1981:65–72.
32. Gillespie JM. The structural proteins of hair: isolation, characterisation and
regulation of biosynthesis. In: Goldsmith LA, ed. Physiology, Biochemistry and
Molecular Biology of the Skin. Oxford: Oxford University Press, 1991: 625–659.
33. Harding HWJ, Rogers GE. Isolation of peptides containing citrulline and the cross-
link, ( -glutamyl) lysine, from hair medulla protein. Biochim Biophys Acta 1976;
427:315–324.
34. Jones LN, HorrTJ, Kaplin IJ. Formation of surface membranes in developing
mammalian hair fibers. Micron 1994; 25:589–595.
35. Jones LN. Composition and distribution of keratin proteins in human anagen hair
follicles. Br J Dermatol 1994; 134:649–656.
36. Peet DJ, Wetenhall REH, Rivett DE. A comparative study of covalently bound
fatty acids in keratinized tissues. Comp Biochem Physiol 1992; 102B: 363– 366.
37. Peet DJ. Protein-bound fatty acids in mammalian hair fibers. Thesis. Melbourne:
The Russell Grimwade School of Biochemistry, The University of Melbourne,
1994.
38. Birbeck MSC, Mercer EH. The electron microscopy of the human hair follicle. J
Biophys Biochem Cytol 1957; 3:202–233.
39. Rogers GE. Electron microscopy of wool. J Ultrastruct Res 1959; 2:309–330.
40. Forslind B. The growing anagen hair. In: Orfanos CE, Happle R, eds. Hair and
Hair Diseases. Berlin: Springer-Verlag, 1990:73–97.
41. Forslind B, Swanbeck G. Keratin formation in the hair follicle. I: An ultrastructural
investigation. Exp Cell Res 1966; 43:91–209.
42. Parrakkal PF (1969) The fine structure of the anagen hair follicle of the mouse. In:
Montagna W, Dobson RL, eds. Advances in Biology of Skin. vol 9. Hair Growth.
Oxford: Pergamon Press, 1969:441–469.
43. Orfanos C, Montagna W, Stuttgen R. Hair Research. Berlin: Springer-Verlag, 1981.
44. van Neste D, Randall VA, Baden H, Ogawa H, Oliver R. Hair research for the next
millennium. Amsterdam: Elsevier, 1996.
12
The Hair Fiber Surface
Leslie N.Jones
CSIRO Textile and Fibre Technology, Belmont, Victoria, Australia
Mammalian hair fibers are complex chemical and cellular composites, which
usually consist of cell types variously named cuticle, cortical, and medullary.
Cortical and medullary cells comprise the internal regions of the fibers and have
been the subject of intensive research over many decades. Likewise the cuticle,
which makes up the hair fiber surface, has more recently become the focus of
research activities owing to its important role in determining textile and cosmetic
properties. In addition, the cuticle and the surface are involved in various
inherited and nutritional defects, making them important markers of health and
disease.
This chapter will focus on the fiber cuticle and its associated surface
membranes. An explanation of surface properties and functions requires detailed
knowledge of cell structure, ultrastructure, chemical composition, and biology of
formation; hence these studies have formed the basis of our current
understanding. The various studies and the key findings of recent developments
in this important area of fiber science are described.
1
BACKGROUND READING AND METHODS
Many studies relating to fiber structure and properties can be found in
the published proceedings of quinquennial international wool textile research
conferences [1–8]. The early wool studies provided much of our basic
knowledge of the fiber cuticle, and references to various books, reviews, and so
on can be found in Crewther et al. [9] and Bradbury [10]. A major book,
published in 1972 by Fraser et al. [11], describes the composition, structure, and
biosynthesis of mammalian fibers. Later reference books include those by
Asquith [12], Maclaren and Milligan [13], and Leeder [14]. Reviews by Leeder
[15] and Rivett [16] discuss the fiber surface and cell membranes of the wool
fiber. More recent reviews of fiber structure and biology include those of Orwin
[17] Rippon [18], and Jones et al. [19]. New developments in the last decade
concerning fiber surface structure and composition, as well as fatty acid and
lipoprotein compositions, have been reviewed by Jones and Rivett [20], Swift
[21], and Jones [22].
264 JONES
Current knowledge of cuticle, surface structure, and ultrastructure (cell

biology) has resulted mainly from the application of physical techniques such as
optical, scanning, and transmission electron microscopy (SEM, TEM) X-ray
photoelectron spectroscopy (XPS), secondary ion mass spectroscopy (SIMS),
and infrared (IR) spectroscopy. Chemical data have been derived from the use of
biochemical techniques including polyacrylamide gel electrophoresis (PAGE),
amino acid analysis (AAA), and high-performance liquid chromatography
(HPLC). Together, these physical and chemical techniques have provided us
with knowledge of proteins and lipids, their structure, composition, and
interactions.
External surface features of fibers such as contours, defects and damage,
chemical treatments, and polymer coatings are normally observed by means of
SEM. Internal cell structure and ultrastructure including the biology of fiber
formation are studied by usingTEM. Recent developments in atomic force
microscopy (AFM) should expand the potential for even higher resolution
studies of surface characteristics at the atomic and molecular levels. These
combined with the other physical methods (XPS and SIMS) provide a
complementary array of techniques for detailed surface studies.
2
THE FIBER CUTICLE
2.1
Properties and Function
The surface properties of mammalian fibers result essentially from the unique
characteristics of flattened cuticle cells located at their surfaces. These cells
overlap both longitudinally and circumferentially, with the exposed surfaces of
overlapping mouthlike lips or scale edges pointing toward the distal end of the
fiber. The scales are thought to aid in the removal of dirt and vegetable matter. It
has also been suggested that they may assist in anchoring the fiber to the skin
[23]. Cuticle cells are also important in a variety of industrial applications, and
many examples of their properties are exploited in textile processing and in the
cosmetic industry. In the natural world cuticle cells function by protecting fiber
components from environmental damage.
A typical characteristic of mammalian fibers is their high degree of variability.
Features such as morphology, dimensions, and surface properties (luster, texture)
of cuticle cells contribute to this variation. Examples of these features can be
seen in Figure 1, which presents SEM views of fibers from some selected
mammalian species. The variations in characteristics such as surface contours,
scale edges, cuticle thickness, and fiber diameter are notable features. Other
studies show that cuticle thickness varies markedly among species. Pig bristle, for
example, may range from 10 cell layers to 30 cell layers compared with 3 to 7
THE HAIR FIBER SURFACE 265
cell layers in human hair, while in contrast, fine Merino wool fibers possess a
cuticle one cell thick.
Cuticle cell dimensions are also variable among species. In human hair, cuticle
cells are approximately 0.5 µm thick and 60 µm square [21]. In fine Merino
wools the dimensions are approximately 20 µm x 30 µm × 0.7 µm [24]. The
arrangement of cuticle cells in fibers is such that a part of each cell is in contact
with the cortex. Cuticle cells form a circumferential sheath with cells abutting
each other with a general tendency to overlap, thereby forming an exposed edge
usually referred to as the scale edge. The overlapping of cuticle cells in the
vicinity of the scale edges is likely to play important roles in assisting the control
of water ingress and egress. The control of water balance in the internal
structural components of fibers (intermediate filaments/matrix) is an important
part of maintaining normal fiber function and durability [20–22].
Scale edges are the obvious features of fiber surfaces when hairs from different
species are observed by means of SEM.These structures determine
characteristics such as scale heights, which in turn contribute to the surface
properties. It is generally believed that scale height plays a role in tactile
sensations of hairs and textile fabrics. Other observations however suggest that
this issue is probably more complex, and factors such as surface lubricants and
surface chemical composition may also be important in determining these
sensations.
2.2
Cellular Features and Ultrastructure
Detailed knowledge of the fine internal structure of cuticle cells has resulted from
many decades of studies using transmission electron microscopy (TEM). When
ultrathin sections are positively stained and examined by TEM (Fig. 2), the cuticle
ultrastructure is seen to consist of a laminated arrangement of variously stained
components [11, 25, 26]. At the junction between the embedding medium and
the outermost region of the cuticle, a thin band can sometimes be visualized, but
only after the application of special staining procedures [27] or after the
application of techniques for coating the surface of this band [28]. This surface
band has usually been referred to as the epicuticle [11] or, more recently, the
fiber cuticle surface membrane [20]. The densely stained band below this surface
membrane is known as the A layer, which together with an underlying broader,
less stained band forms the so-called exocuticle [26]. The innermost unstained
band forming the remainder of the cuticle cell is known as the endocuticle [17, 26].
The structural characteristics of each of these layers are described in
Sections 2.2.1 to 2.2.3.
266 JONES
FIGURE 1 Micrographs of some mammalian fibers obtained with the scanning electron
microscope: (A) human, (B) dog, (C) cat, (D) merino wool. The surface textures
demonstrate marked variability in features such as cuticle cell arrangements, cuticle scale
edges and surface contours.
2.2.1
Fiber Cuticle Surface Membrane (Epicuticle)
Estimates of the surface membrane depth have been the subject of many studies,
FIGURE 2 Transmission electron micrograph (transverse section) of human hair fiber

cuticle (FCU) obtained after bulk staining with osmium tetroxide and post-staining with
uranyl acetate and lead citrate. Each FCu cell comprises an outer exocuticle (exo), which
contains the densely stained A layer. Below the exocuticle the endocuticle occupies the
remainder of the cell and contains various cellular remnants (cr). The intercellular regions
between FCu cells make up the cell membrane complex (cmc). Bar=0.1 µm. (From Ref. 20.)
and a definitive dimension has yet to be established. Despite the wide range of
estimates, it is generally agreed that the thickness of this surface layer is
between 2 and 7 nm [29–31]. Yet despite the relatively thin dimensions of this
surface membrane, the complexity of structure and composition together with its
important role in wettability, friction, surface tension, and tactile properties
warrant further detailed studies. In many respects the fiber cuticle surface
membrane (Fig. 3) should be considered in terms of the plasma membranes from
which it is derived. The formation of the surface as part of fiber cuticle cell
development is discussed in more detail later.
In the mature fiber, the cuticle surface membrane contains a lipid and a
proteinaceous component, and in this respect it shows differences from the plasma
membranes of follicle cells. Hence important modifications take place during the
formation of a specialized cuticle cell surface in follicle cells. The most
significant difference is that the lipid moiety at the fiber surface almost certainly
consists of monolayers in contrast to the bilayers found in plasma membranes.
As stated earlier, “epicuticle,” is sometimes used to designate the surface
membrane, but use of this term presents complications. Among these is the
original definition of the epicuticle [32] as “the membrane raised from the fiber
surface after treatment with chlorine and water” (Fig. 4). Essentially this process
liberates a proteinaceous residue free of lipids. Therein lies the significant and
important difference between the surface membrane in situ (lipoprotein) and the
isolated epicuticle residue (protein). Hence use of “epicuticle” to refer to the
surface membrane in situ cannot be correct.
To put the lipid and protein components of the surface membrane into context,
it is convenient to consider the properties of the lipids and the proteins as
268 JONES
FIGURE 3 Transmlssion electron micrograph at high magnification (transverse section)

showing the fiber cuticle surface membrane (FCUSM) in association with the other
cuticle components. The fiber was stained with osmium tetroxide and the section with
potassium phosphotungstate to reveal the FCUSM as a thin, lightly stained band at the
surface above the A layer (a) of the exocuticle (exo). The FCUSM is approximately 6 µm
thick and contains fatty acids including 18-methyleicosanoic acid and proteins stabilized
by isopeptide cross-links. Bar=0.1 µm. (From Ref. 20.)
separate entities. However, since these two moieties are chemically (covalently)
linked, a discussion of the nature of this linkage is also included.
2.2.2
Fiber Cuticle Surface Lipids
The lipids existing at the outermost surface of mammalian fibers are arguably the
most important components of the surface, since they form a hydrophobic
interface between the environment and the fiber. In this capacity the surface
lipids function as the first component at the various interfaces encountered
during fiber interaction (e.g., fiber-air, fiber-water).
To explain the hydrophobic nature of the fiber cuticle surface membrane and
the role of lipids in this “membrane,” it has been suggested that these lipid
moieties are covalently bound to an underlying protein matrix [33, 34]. A later
study by Kopke and Nilssen [35] postulated that the surface of the fiber cuticle is
FIGURE 4 Micrograph of a wool fiber after treatment in a mixture of chlorine and water.
The “bubbles” raised at the fiber surface are enclosed by a thin proteinaceous membrane
known as the epicuticle. The reaction with chlorine removes surface fatty acids and
imparts hydrophilic properties to the residual epicuticle.
studded with long-chain fatty acids linked by esters to underlying proteins. These
authors reasoned that the existence of ester bonds would account for the effect
observed after treatment of fibers in alkaline solutions. In a more recent study by
Leeder and Rippon [36], wool fibers treated with anhydrous potassium tertiary
butoxide in the nonswelling solvent tertiary butanol were combined to give a
reaction presumed to be confined to the fiber surface. After the reaction, the
authors observed dramatic increase in the fibers’ wettability properties
(hydrophilicity), which was subsequently attributed to the removal of fatty acids
at the exposed fiber surface. Subsequently the authors introduced the term F
layer to describe the presence of fatty acids on the fiber surface.
Analysis of the fatty acids removed by alkaline treatment (potassium tertiary
butoxide in tertiary butanol) revealed that the major lipid component (58% of
total fatty acids) was a methyl branched 21-carbon fatty acid [37].
Further investigation confirmed the presence in wool fibers of a class of lipids
that were resistant to extraction with lipid solvents but were liberated by mild
alcoholic alkali treatments. Total alkali digests of wool that had been pretreated
to remove all the extractable lipids liberated predominantly fatty acids (0.8–1.3
mg per gram of fiber) [38–41]. The composition of the fatty acids is presented in
Table 1. From the separate analyses, the 21carbon branched-chain fatty acid was
found to comprise about half the total bound fatty acids. This 21-carbon fatty
acid was also present in the bound lipids of human hair and was identified mass
spectrometrically as 18methyleicosanoic acid (MEA) [42, 43]. A more definitive
270 JONES
identification of MEA in these digests isolated from wool fibers has been made
by Negri et al. [41], who used NMR and mass spectrometry. The estimate of the
amount
TABLE 1 Major Fatty Acids in Fiber Bound Lipidsa

Composition (wt%)
Fatty acid Woolb Merino woolc Human haird
16:0 11 8 17
18:0 12 6 10
18:1 8 5 5
21:0a 43 72 48
aBound fatty acids liberated from solvent extracted wool (alcoholic alkali).
bLogan et al. [43].
cNegrietal. [41].
dWertz and Downing [44].
Source: Ref. 20.
of bound lipid in wool fibers published by Wertz and Downing [44] was
markedly higher than other published values, with cholesterol sulfate,
cholesterol, and ceramides being significant components.
The total amounts and compositions of the bound fatty acids vary with fiber
types and fiber diameters. Negri et al. [45] showed that an increase in fiber
diameter correlates with a decrease in total bound fatty acids, suggesting that
these bound fatty acids are associated with the surface. Negri et al. [46] also
reported that for a single cuticle cell fiber (e.g., Merino wool), increases in fiber
diameter and the amount of bound MEA as a proportion of total bound fatty
acids decrease in a linear relationship.
Branched-chain fatty acids are frequently found in biological systems but are
usually present as minor components in mixtures of straight-chain saturated and
unsaturated fatty acids [47–51]. It must be noted that the exceptionally high
proportion of a single branched-chain fatty acid as found in mammalian fibers is
very exceptional, and hence MEA would be expected to play a significant role in
determination of fiber surface properties and function.
The function of the MEA is uncertain, especially when its unusual occurrence
is considered together with the additional energy required by the cell to produce
such a unique branched chain, rather than an abundance of the straight-chain
fatty acids (palmitic, stearic, oleic).
To date a number of possible functions have been put forward, including the
following:
A monolayer may be maintained on the surface through inhibition of bi

layer (micelle) formation due to the presence of the branched chain [20].
With the methyl branch located at the hydrophobic end (surface) of the
fatty acid molecule, an umbrella effect, resulting in greater hydrophobicity,
may be achieved [20].
The existence of the methyl branch may render the hydrophobic layer
more resistant to biological degradation [20].
The layer of 18-MEA at the surface of undamaged hair has a strong
influence on the frictional behavior of the hair [21].
Despite the amount of data accumulated on the biological distribution of 18-

methyleicosanoic acid and its specific localization in the hydrophobic barrier
layers of mammalian hair fiber cuticle cells, more research is needed to
determine the distribution of 18-MEA throughout the boundary layers of the
cuticle cells.
Most workers now generally agree that the surface of mammalian fibers
consists of long-chain fatty acids covalently linked as thioesters to a heavily
cross-linked protein membrane, forming a hydrophobic barrier at the surface of
each cuticle cell. The culmination of chemical research and observations through
electron microscopy has led to the development of a model of a cuticle cell
envelope [20, 45] as demonstrated in Figure 5. In support of this hypothesis,
studies using static secondary ion mass spectrometry (SSIMS) and X-ray
photoelectron spectroscopy (XPS) have been undertaken [52–54]. Ward et al.
[54] used atomic ratios from XPS and estimated the depth of the surface lipid
layer as 0.9 nm, which is less than half the figure calculated by Negri et al. [45]
from the length of a 20-carbon chain. In attempting to account for this
discrepancy, Zahn et al. [55] suggested that the anomaly could be accommodated
if the fatty acids are folded back in the direction of the surface. Peet et al. [56]
suggested that the apparent contradiction in the thickness of the lipid layer may
be an art-fact of the anhydrous, high-vacuum conditions used in the XPS studies.
This suggestion that the fiber surface could differ in vacuum conditions and in
other environments such as in air or liquids may be important.
In 1997 Horr [57] analyzed the results of studies of contact angles and surface
energy values determined for wool fibers [58] as these data relate to surface
components such as methyl, methylene, keratin, and absorbed vapors. Horr
concluded that the outermost region of the fiber does not consist of methyl groups
exclusively, as proposed in the model of Negri et al. [45]. It must be remembered
the method used to measure the contact angle must include the regions (scale
edges, etc.) between cuticle cells in addition to the cuticle cell itself. Hence
vapor adsorption due to capillary condensation may occur at the fiber cuticle
scale edges, potentially contributing to the interpretation by Horr that the fiber
surface is not entirely methyl.
More recent studies of the fiber surface have utilized new developments in
scanning probe microscopy (SPM). Atomic force microscopy (AFM), a form of
SPM developed in 1986, is used to obtain information about the surface
topography and nanomechanical properties of a material [59]. The instrument
272 JONES
FIGURE 5 The current model proposed for the arrangement of proteins and lipids at the
surface of the fiber cuticle consists of a monolayer of fatty acids substantially containing
18-methyleicosanoic covalently linked through thioester bonding to an underlying
proteinaceous matrix. The protein component is essentially equivalent to the so-called
epicuticle residue obtained after treatment of fibers with chlorine-water mixtures. This
protein moiety is also thought to contain a network of isopeptide bonds for increased
stabilization, which distinguishes it from the A layer of the exocuticle. (From Ref. 20.)
itself, which can be operated under a range of environmental conditions (under

vacuum, in air, under liquids), is therefore an ideal tool for examining the effects
of various treatments and environmental conditions on the surface of hair fibers.
Several workers have used AFM to examine the morphology of the surface of
human hair and have revealed fine structures such as the exocuticle, the
endocuticle, and the A layer [60]. Swift and Smith [61] showed that striations
cover the entire outer cuticular surfaces of undamaged fibers, except at the
margins of the overlapping scales. Further AFM studies have found that these
striations are common on all mammalian keratin fibers (including those from
montremes); moreover, when covalently bound fatty acids are removed from the
surface, striations are still present on the residual surface [62]. This supports the
view that the striations arise following contact with the inner root sheath in the
follicle [62].
The effects of various conditions and treatments on the surface structure have
been revealed by using various modes of AFM. For example, topographical
images have shown the effects of hydration [60, 63–65], pH [65, 66],
temperature [66], conditioners [60, 64] cationic polymers [67] and removal of
the covalently bound lipid layer [62, 63], while lateral force mode has been used
to examine variations in structural and frictional properties of fibers that have

received various treatments [60, 68].
More recently, the force-distance mode has been used to examine the
adhesional properties of 18-MEA [69] and the surface of fibers that have been
exposed to water and shampoo and conditioner [63]. The thickness and stiffness
of the lipid layer have also been calculated by using force– distance mode [63].
Blach et al. [64] estimate the lipid layer to be 7.6 to 30 µm thick, with an
effective Young’s modulus of 1.2 to 8.4 MPa. This result is in strong contrast to
current concepts of the lipid layer and is in need of further clarification.
2.2.3
Fiber Cuticle Surface Protein
To sustain thioester bonding between the surface monolayer of fatty acids, an
underlying supporting protein matrix must exist such that available cysteine
residues are presented to the outer surface. Negri et al. [45] suggested that this
protein matrix may contain -pleated sheet in which protein chains lie parallel
and are linked through hydrogen bonding. In this conformation amino acid side
chains would be at regular intervals along each sheet. The presence of amino
acid side chains on the other side of the sheet at the same density indicates that a
relatively moderate number of cysteine residues could provide the required
bonding with surface fatty acids [21]. It is generally agreed that an overwhelming
need exists for research to isolate and analyze protein sections with bound lipids
intact for sequence studies to address the many unanswered questions
surrounding this hair surface composite envelope. Such studies could make a
range of data available including protein sequence determination, protein links,
gene identification and protein conformation.
Significant progress has been made in studies of the cornified envelope in
formation of epidermal stratum corneum. Current ideas suggest that soluble
protein precursors such as involucrin (an elongated structural protein) and
cystatin and elafin (protease inhibitors) are initially deposited and cross-linked
by the action of a transglutaminase to form a scaffold structure. Other proteins
such as loricrin and various members of the family of small proline-rich proteins
are attached presumably through the action of a second transglutaminase. These
complexes then form the protein portion of the cornified envelope to which -
hydroxyceramides bearing fatty alcohols and sphingosine chains of variable
length become attached [70].
Despite the obvious variations in composition that may exist between hair and
stratum corneum, it is conceivable that similar events occur in the formation of
the hair fiber surface and even possibly in nails. Evidence for the existence of
involucrin has been found indirectly from studies of amino acid analyses [71].
The current data supporting similarity between the hair cuticle surface proteins
and the cornified envelope in stratum corneum can be summarized as follows:
274 JONES
The presence of high contents of isopeptide cross-links leads to high resistance

of keratolytic agents [72–75].
Epicuticle preparations were shown by Bindewald in 1983 to contain loricrin
and involucrin (Zahn, personal communication).
Resistant membranes of hair fibers contain small proline-rich proteins [71].
3
EXOCUTICLE (PROTEINACEOUS RESISTANT
BARRIERS)
The regions below the proteolipid complexes of the fiber cuticle surface
membrane are known as the exocuticle. In positively stained sections examined
by TEM, an outer densely stained band of the exocuticle is known as the A
layer; the remaining exocuticle being less intensely stained (Fig. 2). These layers
presumably act as resistant barriers but should not be confused with the resistant
membranous residues obtained following fiber degradation in strong acids and
other destructive agents [15]. In the preparation of resistant membranes, it is
expected that the exocuticle must have dissolved almost completely. From this
assumption, it can be suggested that the sulfur-rich exocuticular proteins are
devoid of the isopeptide bonds existing in the resistant membranous residues.
The A layer is a predominantly proteinaceous component with a fairly uniform
thickness, estimated in human hair as approximately 100 nm [21]. Since the A
layer has a high cystine content (1 in every 2.7 residues as half cystine) [76], it
can be deduced that certain members of the ultrahigh-sulfur proteins are located
in it. Evidence exists that part of the A layer is resistant to digestion by mixtures
of papain and reducing agents (dithiothreitol) that dissolve most other fiber
components [77]. It is also conceivable that the actions of transglutaminases in
the surface membranes extend their effects partly into the underlying A layer to
catalyze the formation of isopeptide bonds [ -amino-( -glutamyl)-lysine]. Several
studies [78, 79] have found various levels in enzyme-resistant material that are
presumed to be derived from the A layer. Since the available evidence suggests
these isopeptide bonds are located in the upper regions of the A layer, it would
appear that these layers determine the origins of the epicuticle membranes obtained
after chlorine-water treatment.
The majority of cysteine residues in the A layer are involved in formation of
intermolecular disulfide bonding [80], where they act as the proteinaceous
barriers that protect the fiber from mechanical, chemical, and biological attack. It
seems that the A layer may also provide support for the rigid attachment of
covalently linked fatty acids at the fiber surface, a function that in turn assists
with establishing the frictional properties at the surface.
The remainder of the exocuticle appears to be generally amorphous in the
TEM. It is predominantly proteinaceous and lies below the A layer with variable
thickness (100–300 nm in human hair) [21]. Developments in micro-analysis by
means of TEM [81–83] have provided direct evidence to show the location and
relative distribution of sulfur-rich regions in the endocuticular (low-sulfur)

exocuticular (sulfur-rich) and A layers (very sulfur-rich) of the cuticle (Fig. 6). In
support of this finding, electron histochemical studies have shown that the A
layer has higher sulfur content than the exocuticle [84].
When exocuticle is exposed to treatment with papain-dithiothreitol, dissolution
is relatively rapid, indicating that this layer is devoid of isopeptide bonds [77].
To date both the exact nature of the exocuticle and how many proteins are
located in it are unclear but, recent developments in transgenic wool fibers have
indicated that the KAP 5 [85] ultrahigh-sulfur proteins are an important
component of this layer. It is expected that numerous other ultrahigh-sulfur
proteins identified in polacrylamide gel electrophoretic (PAGE) studies of fiber
protein extracts must also reside in the exocuticle, but the locations of these
proteins remain to be directly demonstrated. An example of a two-dimensional
PAGE study of a wool extract showing the major classes of proteins is presented
in Figure 7. The available evidence indicates that the exocuticle contains high
cystine levels and hence high concentrations of disulfide bonding. Given these
characteristics, it is expected that the lower exocuticular layer also provides a
proteinaceous barrier to assist in fiber protection from environmental damage
while perhaps being less rigid than the A layer.
4
ENDOCUTICLE (FIBER CUTICLE CUSHION)
The so-called endocuticle occupies the inner region of the fiber cuticle and is
located below the exocuticle (Fig. 2). In TEM observations the endocuticle is
revealed to be low in electron density and variable in thickness (human hair, 50–
300 nm) [21]. Analytical TEM studies show markedly lower sulfur contents
(Fig. 6) than the exocuticular layers [81–83]. The appearance of the endocuticle
texture has led researchers to suggest that it is derived from the developing cell
cytoplasm and cytoplasmic components (nucleus, cell organelles). The high
susceptibility of this endocuticle to proteolytic enzymes [86] indicates that it is
one of the weakest and most accessible parts of the fiber. Apart from the
distinctly characteristic organelle remnants, the endocuticle is generally
amorphous and little is known about its protein composition.
In textile processing the endocuticle is considered to be highly accessible to
dyes and other reagents. This characteristic has been demonstrated in analytical
TEM studies [87], which clearly demonstrated that dyes were preferentially
located in the endocuticle. It should be noted that the mechanisms by which dyes
enter the fiber are still equivocal because it is difficult to study a dynamic
process by means of static techniques such as microscopy.
The endocuticle has been found to contain high levels of acidic and basic
amino acids and is also apparently devoid of isopeptide bonds [21]. Taken
together, these features support the view that the endocuticle is mechanically
“soft” and has a high capacity to swell in water, in contrast to the rigid region of
276 JONES
FIGURE 6 Elemental sulfur X-ray maps obtained from transverse sections of fiber
cuticle cells (merino wool) show the relative distributions of sulfur (A) in the exocuticle
and the endocuticle. The outermost A layer of the exocuticle is apparently richer in sulfur
than the remainder of exocuticle. The endocuticle appears to have a relatively low sulfur
content. (B) The bright field (scanning TEM image) of the same section, included for
comparison. Bar=0.1 µm. (From Ref. 20.)
the exocuticle. These observations have been supported by recent studies using
AFM before and after immersion of fibers in water [21].
The endocuticle is apparently an important region for consideration in aspects
of fiber modification and processing. Its relative reactivity and accessibility
make it significant for developing processes involving reagent penetration. In
nature it may appear to be relatively unimportant, although it is tempting to
speculate that its primary function has been adapted to provide a “cushion”
between external environmental forces and the underlying cortical cell regions
that comprise the main bulk of the fiber.
FIGURE 7 Fluorograph of wool fiber proteins separated into the various major classes
using two-dimensional polyacrylamide gel electrophoresis. The lowsulfur proteins are
located in the hard -keratin intermediate filaments (KIF) of the cortex. High-sulfur and
high-glycine/tyrosine proteins are mostly associated with KIF in the cortex, while the
ultrahigh-sulfur proteins are thought to be predominantly located in the exocuticle region
of the fiber cuticle.
5
SEPARATION OF FIBER CUTICLE CELLS
Various methods for isolating fiber cuticle cells have been developed. Ley and
Crewther [88] used alkaline reduction for extraction of cell polypeptides from
isolated cuticle cells. The low solubility of proteins (30% w/w) supports the
evidence suggesting the presence of isopeptide bonds in the exocuticular layers as
discussed earlier. In amino acid and gel electrophoresis analysis the isolated
polypeptides were rich in cystine and comprised a clearly distinct class of
proteins [88].
278 JONES
5.1
Intercellular Regions (Cell Membrane Complex)
It is important to consider the various membranes and inter-intracellular regions
associated with cellular contact and communication between the fber cuticle and
the cortex. In particular, the lipid-containing plasma membranes found in the
follicle play an important role in the formation of the surface and intercellular
contacts and should be considered in relative detail (see Sec. 2). A discussion of
the cell membrane complex is also justified by considering it to be part of a
multilayer of cuticle cells like those found in human hair. As outwardly exposed
cuticle cells are removed by various processes, cleavage often occurs at the
intercellular regions of the cell membrane composition to expose a new fiber
surface. Clearly this cleavage process and the properties of the components in the
cell membrane complex need to be understood.
The intercellular connections between cuticle and cortical cells of mammalian
fibers are characterized by densely stained intercellular bands ( layers)
sandwiched between two nonstaining thinner bands ( layers) (Fig. 8) [11, 15].
Based on the definition of Leeder [15], these three bands normally comprise the
cell membrane complex. An additional densely stained band associated with the
intracellular membrane and known as the I layer has been suggested by some
workers [11] to be part of the cell membrane complex. Despite this sulfur-rich I
layer forming an intracellular band that may be considered to be part of the cell,
it should be emphasized that the layer may play an important role in the
stabilization of the cell membrane complex.
5.2
Cell Membrane Complex Components
5.2.1
Delta Layers ( )
The generally accepted dimensions of the cell membrane complex are in the
range 25 to 28 nm, with the centrally located, densely stained band ( layer)
being approximately 15 to 18 µm thick. It was shown in 1997 [20] that the
layer contains a series of lamina with a central lighter stained band
approximately 5 µm thick. Some difficulty has been encountered in determining
the precise composition of the layer owing to the difficulty of isolating it
intact. It is generally agreed, however, from studies using histochemistry [21]
and microanalysis (Jones unpublished), that the sulfur content is relatively low.
The presence of protein in the layer is also uncertain. Evidence from enzyme
studies [77] suggests that carbohydrate and histochemistry [89] or mixtures of
proteins and polysaccharides may exist. Intense staining of the layer with
phosphotungstic acid also supports the existence of polysaccharides. Other work
by Orwin [90] also demonstrated polysaccharides on the surface of presumptive
hair cells in the follicle. Hence it would appear from the available evidence that
the polysaccharides play a significant role in the processes of cell adhesion,
together with any other functional roles performed by the layer in the cell
membrane complex.
5.2.2
Fiber Cuticle Cell Membranes ( layers)
The unstained bands of the cell membrane complex observed by transmission
electron microscopy are normally referred to as the layers [26]. These layers
should be considered more correctly as modified plasma membranes, since
considerable changes occur in their structure and composition as they pass from
follicle to fiber. The layers (5 nm thick) are usually unstained in the mature
fiber, while in the follicle the unstained region is thinner and a stained outer band
is clearly evident [91]. This apparent difference would seem to reflect significant
differences in composition. One possible explanation is that polar groups such
as phosphate moieties, which are part of the phospholipid components of plasma
membranes, are degraded during fiber development. Various analyses of fibers
have supported this idea by indicating that fibers are virtually devoid of
phosphorus [72, 17, 20, 72]. In a review such as this, primarily concerned with
the fiber surface, the layers are particularly important owing to the concept that
plasma membranes are the precursors of the layers and in turn part of the
surface. It should also be noted that in fibers with multiple layers of cuticle cells
erosion of a surface cuticle cell exposes a new surface, which in turn comprises a
surface component layer. An important observation evident in micrographs of
human hair is that the nonstaining layer (between the arrows in Fig. 8) at the
surface of the fiber cuticle cell is clearly different from the layer on the
underside. The apparent difference is the greater thickness of the surface layer,
which strongly implies compositional differences in the upper and lower layers.
These observations have initiated a range of studies to arrive at an understanding
of the structure and composition of surface and underside membranes in fiber
cuticle cells. The location of 18-methylei-cosanoic acid was therefore explicable
only after the mechanisms underlying the formation of surface membrane had
been demonstrated by means of high-resolution transmission electron
microscopy with energy filtering in conjunction [92] with other studies of mutant
hairs in patients with maple syrup urine disease. These images demonstrate the
location of 18-methyl-eicosanoic acid as predominantly in the upper or
presumptive surface layers of fiber cuticle cells [93, 94].
Various observations have demonstrated in addition that the surface layers
are laminae of preferred cleavage when fibers are subjected to certain stresses
[21]. Before definitive conclusions can be made in this regard, it will have to be
quantitatively demonstrated that the surface layer is specifically mechanically
weaker than surrounding cuticle components including the lower layers.
280 JONES
FIGURE 8 High-magnification transmission electron micrograph of the cell membrane

complex (cmc) of human hair between two apposed fiber cuticle cells (FCU). The cmc
consists of a pair of unstained modified membranes ( layers) and an intercellular layer
of higher staining intensity. An important feature is that the surface layer (apposed to
the A layer) appears wider than the lower layers of a cuticle cell. The layer has a series
of internal laminations under these conditions of staining. An intracellular
membraneassociated layer (i) forms a narrow band on the underside of a fiber cuticle cell.
The dark-stained band (a) is the surface A layer of the exocuticle (exo). The fiber was
prereduced and stained with osmium tetroxide, and the sections were poststained by using
uranyl acetate and lead citrate. Bar=100 nm. (From Ref. 20.)
6
FIBER CUTICLE CELL DEVELOPMENT
In the follicle, presumptive fiber cuticle cells are aligned with the developing
cortex, where they form a distinct morphological pattern. During their
differention, fiber cuticle cells are flattened and develop an outward slope that is
thought to be derived from relative cell movements and lateral forces generated
by the apposed innermost surface of the inner root sheath cuticle [17]. A unique
intracellular event observed in developing fiber cuticle cells is the formation of
electron-dense granules at an early stage of differentiation. As the process
continues, granule production increases, and the granules appear to fuse to form
a continuous network in the outer portion of the cell cytoplasm. It would appear
that the exocuticle forms (Fig. 9) from the aggregation of these protein granules;
it should be noted, however, that the molecular bases of this migration process
and the conformational changes that occur in the formation of the exocuticle are
virtually unknown.
In a study by Woods and Orwin [95] three types of granules were
distinguished in the formation of fiber cuticle cells. The granules of types I and
II are smaller than the type III forms and were considered by these authors to be
precursors of the A layer. The type II forms were suggested to form the
convoluted laminae of the developing exocuticle.
In a later study, Jones et al. [92] observed that the exocuticular laminae
contained highly electron-dense cores and suggested they may be precursors of
the A layer. This observation is presented in Figure 10, which shows two
apposed fiber cuticle cells at different stages of development. In the cell denoted
B the A layer is clearly delineated, while in the cell marked A the A layer is
absent and the densely stained cores of the exocuticular laminae are present. Hence
it would appear that cell A is a precursor to cell B and that the contents of the
dense lamina cores are the A layer precursors. Concomitant with the formation
of the exocuticle, other differention processes in fiber cuticle cells involve
condensation and dehydration of the inner flattened zone of residual cytoplasm
that goes to form the endocuticle.
7
FORMATION OF THE FIBER CUTICLE SURFACE
MEMBRANE
During the formation of the exocuticle, marked changes also occur in the plasma
membranes and intercellular regions of developing fiber and inner root sheath
cells [96]. Associated with these changes is activity of acid phosphatase in the
intercellular regions between fiber cuticle and inner root sheath cuticle cells. In
addition, polysaccharides in the surface membrane of keratinizing cells have
been observed [90].
Details of the morphological changes in the surface membranes and
intercellular regions have been established by using high-resolution transmission
electron microscopy incorporating energy-filtered imaging [92]. This technique
has enabled investigation of the developing fiber cuticle surface throughout its
formation up to region in which the fiber enters the pilary canal.
282 JONES
Associated with the formation of exocuticular laminae in developing fiber

cuticle cells, the plasma membrane on the outer surface appears to be first
disrupted, then replaced by paired laminae of presumed lipid bilayers, which are
deposited on the fiber cuticle surface and occupy the intercellular regions
between inner root sheath and fiber cuticle cells (Figs. 9 and 10). A densely
stained central band in the laminae forms the cleavage line as inner root sheath
cuticle and fiber cuticle cells separate. After this cleavage process, one pair of
laminae is deposited on the densely stained exocuticle layer, while the other pair
is located on the inner root sheath cuticle surface. The cleavage line continues
around the presumptive scale edge of fiber cuticle cells and extends between two
overlapping cuticle cells to form an overlap of approximately 1 to 2 µm (Fig. 11).
The plasma membranes on the underside of fiber cuticle cells appear to have
maintained their original morphology and staining characteristics throughout the
differentiation process. In these underside regions, intercellular material between
apposed fiber cuticle cells demonstrates an unstained central band sandwiched
between relatively lightly stained single laminae ( layer). A model diagram of
the emerging fiber cuticle cell is shown in Figure 12 .
Given the properties exhibited by the mammalian fiber cuticle surface, it
would seem reasonable to suggest that a new membrane needs to be constructed.
We know that the newly emergent fiber surface is uniquely hydrophobic and
water resistant. It is also paradoxical that the surface membranes must also be
assembled in a way that allows water to enter the internal fiber components. The
overlap region at the scale edge between fiber cuticle cells seems to be an
adaptation that could presumably perform this role [92].
8
MUTATIONS IN THE FIBER CUTICLE SURFACE
MEMBRANE
An inherited defect involving the synthesis of the branched-chain fatty acids such
as 18-methyleicosanoic acid has been described in mammalian species. Maple
syrup urine disease (MSUD), or branched-chain ketoaciduria, has long been
known in humans, and a similar mutation has been recently identified in polled
Hereford cattle [97]. MSUD results from an inborn error in metabolism leading
to a deficiency of the enzyme branched-chain 2-oxo acid dehydrogenase. The
deficiency leads to accumulation of the branched-chain amino acids leucine,
isoleucine, and valine, together with their respective -keto acids in the blood,
urine, and cerebrospinal fluid [98, 99].
FIGURE 9 Transmission electron micrograph (transverse section) of a fine wool follicle cuticle cell. At this high magnification the developing
fiber cuticle cell (FCu) can be seen in apposition to an inner root sheath (IRSCU) cell. In the FCU cell formation of the exocuticle (exo) is
under way. The exo structure appears to be composed of lamellated components that show dual staining. It is notable that the original plasma
membrane is absent and four intercellular laminae occupy the intercellular regional between FCu and IRSCU cells. These intercellular laminae
are thought to contain lipids such as 18-methyleicosanoic acid. In this micrograph, the FCu and IRSCU cells are separating along a central
THE HAIR FIBER SURFACE
cleavage plane in the intercellular laminae. The section was stained with osmium tetroxide, uranyl acetate, and lead citrate. Bar=0.1 µm. (From
Ref. 20.)
283
284 JONES
FIGURE 10 Transmission electron micrography shows two developing fiber cuticle cells (FCu) at different stages of formation in a wool
follicle. In the cell denoted A, the exocuticular lamellae (exo) exhibit densely stained cores, and this cell is a precursor to cell B. In cell B, the
dark-stained core material has migrated to the outer surface region of the cell to form the presumptive A layer of the exocuticle. The separation
of the FCu and IRSCU cells is also evident in the central intercellular region. The specimen was stained with osmium tetroxide, uranyl acetate,
and lead citrate. Bar=0.1 µm.
FIGURE 11 Transmission electron micrograph showing the developing fiber cuticle cell (FCU) at the region of the presumptive scale edge.
The cleavage process between FCU and inner root sheath cuticle (IRSCU) is well advanced and can be seen to extend from between
overlapping cells and around the scale edge to terminate on the underside of the FCU cell, creating a channel between overlapping FCU cells.
THE HAIR FIBER SURFACE
Note also that the exocuticle also terminates in the same region on the underside of the FCU cell. The cleavage between cells occurs between
the apposed FCU and IRSCU cells along a central dark-stained band. The section was stained with osmium tetroxide, uranyl acetate, and lead
285
citrate. Bar=0.1 µm. (From Ref. 20.)

286 JONES
FIGURE 12 Formation of the fiber cuticle surface membrane (FCUSM) and the other
FCU cell components, including the exocuticle (exo) and the cell membrane complex
(CMC). The FCU has separated from the inner root sheath cuticle (IRSC) and is now
entering the pilary canal (PC). The plasma membrane (PM) of the IRSCU is coated with a
pair of intercellular laminae (PIL). The plasma membrane of the FCU cell has been
disrupted and replaced by a pair of intercellular laminae (PIL), and these are in contact
with the A layer (a) of the exocuticle (exo). The endocuticle is donated ends and the CMC
comprises to layers (B) and an intercellular delta layer (5). Fatty acids such as 18-
methyleicosanoic acid are components of the paired intercellular laminae on the FCU
surface (From Ref. 20.)
It is generally believed that anteisomethyl branched-chain fatty acids such as

18-methyleicosanoic acid (MEA) form from 2-methylbutyric acid as a result of
oxidative decarboxylation of isoleucine by branched-chain 2-oxo acid
dehydrogenase [100]. Hence a defect in synthesis of branched-chain 2-oxo acid
dehydrogenase would be expected to eliminate formation of branched-chain fatty
acids. In earlier analyses of hairs from MSUD patients it was found that MEA
was lacking but other straight-chain fatty acids were in the normal ranges [101, 102]
. Hairs from MSUD patients were then found to contain defects associated with
the fiber cuticle surface together with the location and distribution of MEA
[92–94].
In these TEM studies, Jones et al. were able to demonstrate that the structural
defect in MSUD was mostly confined to the surface layers of fiber cuticle
cells. The conclusions from these morphological observations were that the
structural defect associated with the layers correlates with disruptions in the
synthesis of 18-methyleicosanoic acid and that in normal hair this fatty acid is
present in the layers. In addition, since the layers on the fiber cuticle
underside of MSUD hairs appear to be unaffected, it would appear that 18-
methyleicoasnocic acid is confined to the upper surfaces of fiber cuticle layers.
Therefore the upper and lower surfaces of fiber cuticle cells are different with
regard to structure and composition. This conclusion is consistent with earlier
studies by Jones et al. [92], which had shown that a newly derived membrane on
the upper surface and scale edge overlap of fiber cuticle cells had replaced the
original plasma membrane.
Fatty acid analyses of hairs from patients with MSUD and control hairs from
normal individuals have shown differences in the ratios of covalently bound fatty
acids. The most notable difference was the 90% reduction in 18-
methyleicosanoic acid (C21:0a) with a concomitant increase in eicosanoic acids
(C20:0) [94]. Other minor branched-chain fatty acids (C17:0 and C19:0) were
also significantly reduced. Eicosanoic acid is a relatively minor constituent of the
bound fatty acids in normal hair fibers, usually approximately 5%. In the MSUD
hairs eicosanoic acid levels were markedly elevated to become the major fatty
acid (15–19% of the total fatty acids), suggesting that the straight chain form has
replaced 18-methyleicosanoic acid [94], Jones et al. [94] also noted that in
MSUD hairs there appeared to be a general increase in all the linear saturated
fatty acids, particularly C16:0, C18:0, and C20:0, the latter being the most
pronounced. These results contrasted with Naito et al. [102] who concluded that
the deficiency in 18-MEA was not compensated by other fatty acids apart from a
possible small increase in the C20:0.
The functions of 18-methyleicosanoic acid in the surface membrane of
mammalian hair fibers is still not clear, although studies of MSUD hairs indicate
that it plays an important role in intercellular adhesion. Other deductions have
been made about the role of this unusual fatty acid. For example Jones et al. [94]
examined the influence of branched methyl groups in fatty acids and found that
the introduction of a methyl branch lowers the melting point from 77°C
(eicosanoic acid) to 56°C (18-methyleicosanoic acid). Again, given that the
physiological temperature is 37°C, it is not clear whether this difference would
significantly affect the fluidity of the surface membrane. Jones et al. further
suggested that the replacement of C21:0a with C 20:0 in MSUD hairs could
possibly cause greater stiffness or rigidity, possibly leading to a loss of adhesion
between fiber cuticle cells. The studies of MSUD hairs [94] have certainly
indicated that 18-methyleicosanoic acid plays a specific structural or biological
role in the function of fiber cuticle surface membranes. The other findings that
have shown that significant amounts of this uncommon fatty acid are covalently
linked to the hair fiber cuticle of hairs from most mammals also support this
concept [44, 56].
288 JONES
ACKNOWLEDGMENTS
This chapter is dedicated to the memory of the late Dr. D.E. Rivett, who had prior
to his untimely death pioneered many new developments in understanding fiber
surface proteolipid complexes. Many thanks are due to Ms. Jane Sambal for
contributions to AFM developments and to Ms. H. Dixon for preparing the
manuscript.
REFERENCES
1. Crewther WG (ed). Proceedings of First International Wool Textile Research
Conference, vols. A-F, CSIRO, Australia, 1995.
2. Proceedings of Second International Wool Textile Research Conference, Harrogate,
U.K., published as J. Text. Inst. Trans, 51, no. 12, parts 1 and 2, 1960.
3. Proceedings of Third International Wool Textile Research Conference, sections 1–
4, L’lnstitut Textile de France, Paris, 1965.
4. Proceedings of Fourth International Wool Textile Conference, San Francisco,
published as J. Polym Scie Appli Polym Symp 1971, 18 parts 1 and 2,1970.
5. K. Ziegler (ed), Proceedings of Fifth International Wool Textile Research
Conference, vols 1–5, Deutsches Wollforschungsinsitut, Aachen, Germany, 1975.
6. Proceedings of Sixth International Wool Textile Research Conference, vols 1–5,
Pretoria, South Africa, 1980.
7. M. Sakamoto (ed). Proceedings of Seventh International Wool Textile Research
Conference, vols 1–5, The Society of Fiber Science Technology, Tokyo, 1985.
8. GH Crawshaw (ed). Proceedings of Eighth International Wool Textile Research
Conference, vols 1–5, Wool Research Organisation of New Zealand, Christchurch,
1990.
9. Crewther WG, Fraser RDB, Lennox FG, Lindley H. The chemistry of keratins. In
Anfinsen CB, Anson ML, Edsall JT and Richards FM (eds): Advances in Protein
Chemistry. New York, Academic Press, vol 20, 191–346.
10. Bradbury JH, The structure and chemistry of keratins. Adv Protein Chem. 27,
1973, 111–211.
11. Fraser RDB, MacRae TP, Rogers GE. Keratins, Their Composition Structure and
Biosynthesis, Charles C. Thomas, Springfield, IL 1972.
12. Asquith RS. Chemistry of Natural Protein Fibers, Plenum Press, New York, 1977.
13. Maclaren JA, Milligan B. Wool Science, the Chemical Reactivity of the Wool
Fiber. Science Press, Marrackwille NSW, Australia 1981.
14. Leeder JD. Wool, Nature’s Wonder Fiber. Australasian Textiles, Melbourne,
Australia, 1984.
15. Leeder JD. The cell membrane complex and its influence on the properties of the
wool fibre. Wool Sci Rev 1986; 63:3–35.
16. Rivett, DE. Structural lipids of the wool fibre. Wool Sci Rev 1991; 67:1–25.
17. Orwin DFG. The cytology and cytochemistry of the wool follicle. Int Rev Cytol
1979; 160:331–374.
18. Rippon JA. The Structure of Wool, in ed D.M. Lewis. Bradford UK. Wool Dyeing
and Wool Blends, Soc Dyers Colourists 1992, 1–51.
19. Jones LN, Rivett DE, Tucker DJ. Wool and related mammalian fibers. In
Handbook of Fiber Chemistry, Lewin M and Pearce EM (eds), Marcel Dekker,
New York 1998, 355–413.
20. Jones LN, Rivett DE. The role of 18-methyleicosanoic acid in the structure and
formation of mammalian hair fibers. Micron 1997; 28:469–485.
21. Swift JA. Human hair cuticle: biologically conspired to the owner’s advantage. J.
Cosmet. Sci 1999; 50:23–47.
22. Jones LN. Hair structure anatomy and comparative anatomy. In: ed. R. Sinclair,
Dermatological Clinics, Elsevier Science, New York 2001; 50:95–103.
23. Montagna W, Parakkal PF The Structure and Function of the Skin (3rd ed.),
Academic Press, New York 1974.
24. Bradbury JH, Leeder JD. Keratin fibres. IV: Structure of the cuticle. Austr J Biol
Sci 1970; 23:843–854.
25. Orwin DFG. Cytological studies on keratin fibres. In DAD Parry and LK Creamer,
eds, Fibrous Proteins; Scientific Industrial and Medical Aspects, Academic Press,
New York, 1979 Vol 1, 271–297.
26. Rogers GE. Electron microscope studies of hair and wool. Ann NY Acad Sci 1959;
83:378–399.
27. Stapleton I, Jones LN, Holt LA. Interactions between wool weathering and dyeing,
Proceedings of Eighth International Wool Textile Research Conference,
Christchurch, NZ, 4, 117–126, 1990.
28. Mansour P, Jones LN. Morphological changes in wool after solvent extraction and
treatments in hot aqueous solutions. Text Res J 1989, 59, 530–535.
29. Swift JA, Holmes AW. Degradation of human hair by papain. III: Some electron
microscope observations. Text Res J 1965; 35:1014–1019.
30. Lindberg J, Philip B, Gralen N. Occurrence of thin membranes in the structure of
wool. Nature 1948; 162:458–459.
31. King NLR, Bradbury JH. The chemical composition of wool. V: The epicuticle.
Austr J Biol Sci 1968; 21:375–384.
32. von Allwörden K. Die Eigenschaften der Schafwolle und eine neue untersuchungs
methode zum nachweis geschädigter Wolle auf chemischen Wege. Z Angew Chem
1916, 77–78.
33. Elliot RL, Manogue B. An electron microscope study of the surface structure of
wool. J Soc Dyers Colourists 1952; 68:12–14.
34. Lindberg J. Relationship between various properties of wool fibers. Text Res J
1953; 23:67–76, 225–236, 573–588.
35. Kopke V, Nilssen B. Wool surface properties and their influence on dye uptake—
microscopical study. J Text Inst 1960; 51: T1398–T1413.
36. Leeder JD, Rippon JA. Changes induced in the properties of wool by specific
epicuticle modification. J Soc Dyers Colourists 1985; 101:11–16.
37. Evans DJ, Leeder JD, Rippon JA, Rivett DE. Separation and analysis of the surface
lipids of the wool fibre. Proceedings of Seventh International Wool Textile
Research Conference 1985; 1:135–142.
38. Rivett DE, Logan RI,Tucker DJ, Hudson A. The lipid composition of speciality
animal fibres. Proceedings of First International Symposium of Specialty Animal
Fibres, Deutches Wollforschunginstitut an der Technischen Hochschule, Aachen,
1987; 1:128–136.
290 JONES
39. Logan RI, Rivett DE, Tucker DJ, Hudson AF. Analysis of the intercellular and
membrane lipids of wool and other animal fibres, Text Res J 1989; 59: 109–113.
40. Kalkbrenner U, Körner A, Höcker H, Rivett DE. 1990. Studies on the composition
of the wool cuticle. In ed G.H. Crawshaw, Proceedings of Eighth International
Wool Textile Research Conference, Wool Research Organisation of New Zealand,
Christchurch, Vol 3, 398–407.
41. Negri AP, Cornell JH, Rivett DE. The nature of covalently bound fatty acids in
wool fibres. Austr Agric Res 1991; 42:1285–1292.
42. Wertz PW, Downing DT. Integral lipids of human hair. Lipids, 1988; 23: 878–881.
43. Logan RI, Jones LN, Rivett DE. 1990. Morphological changes in wool fibres after
solvent extraction. In ed G H Crawshaw, Proceedings of the Eighth International
Wool Textile Research Conference, Wool Research Organisation of New Zealand,
Christchurch. Vol 1408–418.
44. Wertz PW, Downing DT. Integral lipids of mammalian hair. Comp Biochem
Physio B: Comp Biochem 1989, 92b, 759–761.
45. Negri AP, Cornell HJ, Rivett DE. A model for the surface of keratin fibres. Text
Res J 1993; 63:109–115.
46. Negri AP, Cornell HJ, Rivett DE. The modification of the surface diffusion barrier
of wool. J Soc Dyers Colourists 1993; 63:109–115.
47. Abrahamsson S, Stallberg-Stenhagen S, Stenhagen E. 1963. The higher saturated
branched chain fatty acids. In eds RT Holman and T Malkin, Progress in the
Chemistry of Fats and Other Lipids, Pergamon Press, New York, Vol. 7, 1–164.
48. Ahern DG, Downing DT. Skin lipids of the florida indigo snake. Lipids, 1973; 9:8–
14.
49. Dasguptor A, Ayanoglu E, Djerassi C. Phospholipid studies of marine organisms:
new branched fatty acids from Strongylophora durissima. Lipids 1984; 19:768–
776.
50. Garton GA. Aspects of the chemistry and biochemistry of branched-chain fatty
acids. Chem Ind (Lond) 1985; 9:295–300.
51. Valero-Guillen PL, Martin Luengo F, Larsson L, Jimenez J. Demonstration of 2-
methyl branched-chain fatty acids in some rapidly growing mycobacteria. 1987.
FEMS Microbiol Lett 1987; 44:303–305.
52. George GA, Willis HA, Ward RJ. From aerospace composites to wool: the power
of polymer surface analysis. Chem Aust 1992; 59:56-59.
53. Carr CM, Leaver IH, Hughes AE. X-ray photoelectron spectroscopic study of the
wool fibre surface. Text Res J 1986; 56:457- 461.
54. Ward RJ, Willis HA, George GA, Guise GB, Denning RJ, Evans DE, Short RD.
Surface analysis of wool by X-ray photoelectron spectroscopy and static secondary
ion mass spectrometry. Text Res J, 1993; 63:362–368.
55. Zahn H, Messinger M. Höcker H. Covalently linked fatty acids at the surface of
wool: part of the “cuticle cell envelope.” Text Res J 64, 1994, 554–555.
56. Peet DJ, Wettenhall REH, Rivett DE. The chemistry of the cuticle surface of
keratin fibres. Text Res J 1994; 64:58–59.
57. Horr TJ. A Description of the wool fibre surface based on contact angle
measurements. Text Res J 1997; 67:1–5.
58. Brooks JH, Raman MS. Surface energy of wool. Text Res J 1986; 56:164– 171.
59. Meyer E. Atomic force microscopy. Prog Surf Sci 1992; 41:3–49.
60. Smith JR. Use of atomic force microscopy for high-resolution non-invasive
structural studies of human hair. J Soc Cosmet Chem 1997; 48:199–208.
61. Swift JA, Smith JR. Atomic force microscopy of human hair. 2000, Scanning, 22,
310–318.
62. Swift JA, Smith JR. Microscopical investigations on the epicuticle of mammalian
keratin fibres. J Micros 2001; 204:203–211.
63. Resch R, Ehn R, Tichy H, Friedbacher G. In situ investigation of humidity induced
changes on human hair and antennae of the honey bee, Apis mellifera L. by
scanning force microscopy Appl Phys A, 1998; 66:S607-S611.
64. Blach J, Loughlin W, Watson GS, Myhra S. Surface characterization of human hair
by atomic force microscopy in the imaging and F-d modes. J Cosmet Sci 2001; 23:
165–174.
65. O’Connor SD, Komisarek KL, Baldewschwieler. Atomic force microscopy of
human hair cuticles: a microscopic study of environmental effects on hair of
morphology. J Investi Dermatol 1995; 105:96–99.
66. You H, Yu L. Atomic force microscopy as a tool for study of human hair. Scanning
1997; 19:431–437.
67. Pfau, A, Hossel P, Vogt S, Sander R, Schrepp W. The interaction of cationic
polymers with human hair. Macromol Symp 1998; 126:241–252.
68. McMullen RL, Kelty SP. Investigation of human hair fibres using lateral force
microscopy. Scanning 2001; 5:337–345.
69. Smith JR, Eaton PJ, Waddington K, Alexander C, Tsibouklis J, Swift JA. Adhesion
behaviour of covalently-bound 18-MEA and other fatty acids. Proceedings of Tenth
International Wool Textile Research Conference, Aachen, Germany, HH-2, 1–10,
2000.
70. Jones LN, Steinert PM. Hair keratinization in health and disease. In ed DA Whiting.
Dermatological Clinics. Philadelphia: Saunders 1996, 633–649.
71. Zahn H, Wortmann E-J, Höcker H. Chemical composition of the hair cuticle:
cornified envelope (CE) proteins in epicuticle and A-layer. Proceedings of
European Hair Research Society, Marburg, Germany, Sept. 15–17, 2000, p9.
72. Schwan A, Zahn H. Investigations of the cell membrane complexes in wool and
hair. Proceedings of Sixth International Wool Textile Research Conference,
Pretoria, South Africa, 1980; 2:29–41.
73. Rogers GE. The occurrence of citrulline in structural proteins of the hair follicle. In:
ed. LA Goldsmith, Biochemistry and Physiology of the Skin, Oxford University
Press, New York, Vol. 1, 511–521, 1983.
74. Zettergren JG, Peterson LL, Wuepper KD, Keratolinin. The soluble substrate of
epidermal transglutaminase from human and bovine tissue. Proc Nat Acad Sci USA,
1984; 81:238–242.
75. Banks Schlegel S, Green H. Involucrin synthesis and tissue assembly by
keratinocytes in natural and cultured epithelia. J Cell Biol 1981; 90: 732–737.
76. Swift JA. Minimum depth electron probe X-ray microanalysis as a means for
determining the sulphur content of the human hair surface. Scanning, 1979; 2:83–
88.
77. Swift JA, Bews B. The chemistry of human hair cuticle. 2: The isolation and amino
acid composition of the cell membranes and A-layer. J Soc Cosmet Chem 1974; 25:
355–366.
292 JONES
78. Neinhaus M. Beitrag zur enzymatischen Hydrolyse von Keratinfasern und ihren
Komponenten. PhD Thesis. Rheinisch-Westfälischen Technischen Hochschule,
Aachen, Germany, 1981.
79. Zahn H, Messinger H, Höcker. Covalently linked fatty acids at the surface of wool:
part of the “cuticle cell envelope.” Text Res J. 1994; 64:554–555.
80. Swift JA. The hair surface. In eds CE. Orfanos, W Montagna, G Stüttgen, Hair
Research Springer-Verlag 65–72, 1981.
81. Jones LN, Cholewa M, Kaplin IJ, Legge GJF. Distributions of protein moieties in a-
keratin sections. Proceedings of Eighth International Wool Textile Research
Conference, Christchurch, NZ, 1, 246–255, 1990.
82. Jones LN, Kaplin IJ, Legge GJF. Distributions of protein moieties in a-keratin
sections. J Comput Assist Micros 1993; 5:85–89.
83. Hallegot P, Corcuff P. High-spatial-resolution maps of sulphur from human hair
sections: an EELS study. J Microsc 1993; 172:131–136.
84. Swift JA. Morphology and histochemistry of human hair. In eds P.Jollès, H.Zahn,
H.Höcker, Formation and Structure of Human Hair, Berkhäuser Verlag, Basel,
Switzerland, 149–175, 1997.
85. MacKinnon PJ, Powell BC, Rogers GE. Structure and expression of genes for a
class of cysteine-rich proteins of the cuticle layers of differentiating wool and hair
follicles. J Cell Biol 1990; 111:2587–2600.
86. Swift JA, Bews B. The chemistry of human hair Cuticle. 3: The isolation and
amino acid analysis of various subractions of the cuticle obtained by pronase and
trypsin digestion. J Soc Cosmet Chem 1976; 27:289–300.
87. Sideris V, Leaver IH, Holt LA, Jones LN. Photomodification of the cell membrane
complex of wool—the effect on dyeability. J Soc Dyers Colourists, 1992; 108:436–
440.
88. Ley KF, Crewther WG. The proteins of wool cuticle. Proceedings of Sixth
International Wool Textile Research Conference, Pretoria, South Africa, 2, 13–28,
1980.
89. Swift JA. The histology of keratin fibres. In ed RS Asquith, Chemistry of Natural
Protein Fibers, Plenum Press, New York. 81–146, 1977.
90. Orwin DFG. A polysaccharide containing cell coat on keratinizing cells of the
Romney wool follicle. Aust J Biol Sci 1971; 23:623–635.
91. Fraser RDB, MacRae TP, Rogers GE, Filshie BK. Lipids in keratinized tissues. J
Mol Biol 1963; 7:90–101.
92. Jones LN, HorrTJ, Kaplin IJ. Formation of surface membranes in developing
mammalian hair fibres. Micron, 1994; 24:589–595.
93. Jones LN, Rivett DE. Effects of branched chain 2-oxo acid dehydrogenase
deficiency on hair in maple syrup urine disease. J Invest Dermatol 1995, 104, 688.
94. Jones LN, Peet DJ, Danks DM, Negri AP, Rivett DE. Hairs from patients with maple
syrup urine disease shows a structural defect in the fiber cuticle. J Invest Dermatol
1996; 106:461–464.
95. Woods JL, Orwin DFG. Studies on the surface layers of the wool fibre cuticle. In
eds, DAD Parry, LK Creamer, Fibrous Proteins: Scientific, Medical and Industrial
Aspects. London: Academic Press, Vol. 2, 141–150, 1980.
96. Orwin DFG,Thomson RW. Plasma membrane differentiations of keratinizing cells
of the wool follicle. IV: Further membrane differentiations. J Ultrastruct Res 1973;
45:41–49.
97. Harper P, Dennis JA, Healy PJ, Brown GK. Maple syrup urine disease in calves: a
clinical, pathological and biochemical study. Aust Vet J 1989; 66: 46–49.
98. Zhang B, Healy PJ, Zhao Y, Crabb DW, Harris RA. Premature translation
termination of the pre-Ela subunit of the branched chain a-keto acid dehydrogenase
as a cause of maple syrup urine disease in polled Hereford calves. J Biol Chem
1990; 265:2525–2427.
99. Tanaka K, Rosenberg LE. Disorders of branched chain amino acid and organic
metabolism. In eds WB Stanbury, JB Wyngaarden, DS Frederickson, JL Goldstein,
MK Brown, The Metabolic Basis of Inherited Disease, 5th ed, McGraw-Hill, New
York, 400–473, 1983.
100. Garton GA. Aspects of the chemistry and biochemistry of branched-chain fatty
acids. Chem Ind (Lond), 1985; 9:295–300.
101. Yorimto N, Naito S. Physical and chemical properties of integral lipids in hair cell
membrane complex. Proceedings of International Symposium on Fiber Science and
Technology, Yokohama, Japan, p. 215, 1994.
102. Naito S, Yorimto N, Kuroda Y. The structure of bound lipids of human hair fibres
and its physical properties. Proceedings of Ninth International Wool Textile
Research Conference, Biella, Italy, 1996, 2, 367–374.
13
Biology of Hair Pigmentation
Desmond J.Tobin
University of Bradford, Bradford, United Kingdom
The skin is very important for our health and well-being. It provides a
metabolically active partition separating our internal homeostasis from the
external environment. In this way, the skin protects us from the fluctuating
influences that both threaten and safeguard our health. The skin’s protective
function derives not only from its tough surface epidermal layers, but also from
its associated appendages (e.g., hair follicles, sebaceous gland, sweat glands).
Together these structures continuously, and bidirectionally, transduce
information between our internal and external worlds that trigger, initiate, and
modulate complex life-enhancing response mechanisms.
The skin, given its location, structure, and function, impacts greatly on our
visual appearance. Color is the phenotypic aspect of skin and hair that conveys
more immediate information than any other to the observer. Indeed, nature has
almost outdone herself by providing a phenomenally rich and varied palette of
surface colors that go to highlight the striking superficial variations between
human subgroups and between mammalian species. For hair, these range from
vivid reds and bleached-out blondes to sober browns and raven blacks. Despite
this, hair color per se is derived solely from the pigment melanin synthesized in
specialized organelles called melanosomes. These lysosome-associated packages
of pigment are formed in highly dendritic, neural crest-derived cells called
melanocytes by means of melanogenesis, a phylogenetically ancient biochemical
process.
Although much progress has been made in elucidating the factors involved in
regulating continuous pigmentation in the human epidermis, we are only
beginning to understand the mechanisms involved in regulating pigmentation in
the human hair follicle. This difference is most likely due to the unique cyclical
manner in which hair grows, forcing the construction, deconstruction and
reconstruction of the hair follicle pigmentary unit multiple times during the
average human life. This chapter addresses several critical issues relating to the
biology of hair pigmentation, including the following:
Why human scalp hair is pigmented and then so variably
The development of the hair pigmentary unit
The cell biology of follicular melanocyte subpopulations
296 TOBIN
The biochemistry of melanogenesis

The regulation of hair follicle pigmentation
The remodeling of the follicular melanocyte unit during hair
cycling
The loss of hair pigment during chronological aging
The effect of pigment loss on hair structure
The pathologies of the hair follicle pigmentary unit.
Discussion of several of these issues will rely heavily on murine data, which
are generally assumed to apply to all mammals including humans.
1
THE EVOLUTIONARY SIGNIFICANCE OF HAIR
PIGMENTATION
The particular mammalian trait of hair growth provided mammals with several
critical functions to facilitate evolutionary success, including thermal insulation,
social and sexual communication (involving visual stimuli, odorant dispersal,
etc.), and sensory perception (e.g., whiskers). Hair color provided additional
rewards, including camouflage (e.g., seasonal changes of coat color in the arctic
hare) and social and sexual communication involving visual stimuli (e.g., silver-
backed gorilla). Though it is clear that humans do not need such adaptations for
their actual survival, these traits still play a significant role in our social and sexual
communication. Unlike other mammals, our relative nakedness draws attention
to the hair on our scalp and faces. Unique among primates, human scalp hair can
be very thick, very long, and very pigmented. It is likely that significant selective
pressure was required to retain and exaggerate scalp and facial hair in humans, as
these are potent accompaniments for our already highly expressive faces and are
therefore so critical for optimal communication.
The recent exploitation of molecular genetics has resulted in a flurry of
investigations into the origin of various human subpopulations, and pigmentation
biologists have been at the vanguard of this revolution. While there is an obvious
need for pigment in our weakly haired human epidermis to filter harmful UV
radiation, it is not immediately clear why humans retained and developed such
strong growth of facial and scalp hair. A range of explanations has been
proposed, and it is likely that several of these are indeed functioning in the
retention and development of this human trait. First, a considerable portion of
human evolution occurred littorally, that is, along seacoasts and riverbanks,
where fish was a significant part of people’s diet [1]. Many species of fish
concentrate toxic heavy metals, which will need to be expelled from the body.
These metals can selectively bind to melanin, and hair melanin would be the
favored route of discharge, given the hair follicle’s very high proliferation rate [2].
Long melanized scalp hair could trap or bind chemicals and toxins from heavy
metals, and so prevent access to the living tissue of the highly vascularized scalp.
BIOLOGY OF HAIR PIGMENTATION 297
Moreover, reactive quinone intermediates generated during melanin biosynthesis

exhibit potent antibiotic properties.
It may seem somewhat bizarre that humans living in hot sunny tropical climes
invariably have black scalp hair—not only a trap for radiant heat but also a good
thermal insulator. Clearly, there must have been some benefits that outweighed
these perceived disadvantages. Deeply pigmented skin and hair may protect
against sunstroke, owing to melanin’s very efficient and fast exchange of ion
transport and efflux, which facilitates adequate salt balance [3]. Our assumption
of a bipedal gait positioned our heads for maximum sun exposure, although this
produced its own challenges given that our metabolically expensive brain needs
to be kept cool. The benefit of having the scalp epidermis not only pigmented
but also well-haired may offer cooling effects similar to feather fluffing in birds;
that is, scalp hair could act as a cooling device by keeping the heat away from our
bodies. Moreover, it has been suggested that thick scalp hair, especially when
curly, can generate wind currents much more efficiently than an exposed bald
scalp.
In this context, it is useful to note that hair pigmentation, unlike that in the
epidermis, is not enhanced by UVR (sunlight), most likely because follicular
melanogenic melanocytes are situated below the penetrating depth of UVB
irradiation [4].
Molecular geneticists have started to unravel the enigma that accounts for the
dramatic diversity of hair colors, particularly among northern Europeans. It is
appropriate to note that skin and hair pigmentation phenotypes are linked to
polymorphism at the gene for melanocortin-1 receptor (MCR-1). This receptor is
activated through binding of the proeumelanogenic peptide -melanocyte-
stimulating hormon ( -MSH), an interaction competitively inhibited by agouti
signaling protein (ASP). Most northern European individuals with red hair are
homozygotes or compound heterozygotes for a few MCR-1 mutations [5]. It is
now considered likely that natural selection pressures that ensured dark hair and
skin in the tropics were less critical as humans migrated north, thus permitting
the emergence of loss-of-function mutations in the MCR-1 gene.
2
THE DEVELOPMENT OF THE FOLLICULAR
MELANIN UNIT
Epidermal and hair follicular melanocytes are derived from pluripotent neural
crest cells that commit to the melanocyte lineage. To reach the skin (epidermis
and hair follicles when formed later), so-called melanoblasts migrate along
stereotypical routes. It is a long and eventful journey from the dorsal closing
neural tube, with migration between the dermamyotome of the somites and the
overlying ectoderm until entry into the dermis [6]. Melanocytes are already
present in the human epidermis by 7 weeks estimated gestational age (EGA),
about 2 weeks before hair follicle development begins [7].
298 TOBIN
Much of our knowledge of the development of melanocyte compartments in

the skin and hair follicle derives from the analysis of mutations that effect
differentiation, proliferation, and migration of melanocyte precursors [8]. Of the
more than 90 loci shown to affect hair color in mouse [9], mutations in the
receptor tyrosine kinase ckit [mapped to the white spotting (W) locus in mice]
and its cognate ligand, stem cell factor [SCF; aka mast cell growth factor, Steel
factor and mapped to the Steel (sl) locus in mice] have been most informative.
Mutant homozygotes exhibit an almost complete lack of hair pigmentation, while
ckit mutations in humans are associated with piebaldism [10]. Melanoblast
differentiation itself does not appear to depend on the ckit-SCF interaction, since
melanocyte-specific markers are detectable on these cells before ckit expression.
Similarly, melanoblast migration from the neural crest can begin without the ckit-
SCF interaction. However, if this interaction is lacking, melanoblasts do not
survive [11].
An elegant demonstration of this requirement is the induction of apoptosis in
murine melanocytes after injection of a Kit-blocking antibody (ACK2) during
certain periods of embryonic life [12]. Melanocytes retain this ckit-SCF
requirement also in postnatal life (see later). Moreover, the existence of different
phenotypic outcomes depending on when ckit is antagonized during embryonic
development indicates that these cells pass through ckit-dependent and ckit-
independent stages [13]. Interestingly, melanoblasts that have already entered
developing hair follicles were reported to be resistant to the anti-ckit antibody
and so would appear to be able to survive without a ckit signal. We have recently
shown that murine melanoblasts express ckit as a prerequisite for migration into
the SCF-supplying hair follicle epithelium. Interestingly, fully differentiated ckit-
immunoreactive (ckit-IR) melanocytes were found in the melanogenic bulb,
while non-ckitIR melanoblasts were directed to the outer root sheath and bulge
of fully developed hair follicles [14]. It has been suggested that the oncogene ret
(the receptor for glial cell line-derived neurotrophic factor) can compensate for
the defect of ckit during embryogenesis and postnatal life [15]. Notably, when
ret was introduced into ckit-deficient Wv/Wv mice, high levels of melanin
synthesis were induced in the process of melanocyte development that resulted in
many fully pigmented hairs.
In addition to the ckit-SCF interaction, endothelin 3 and its receptor (Ednrb)
are also essential for melanocyte development [16]. Mice homozygous for an
Ednrb null mutation (piebald-lethal) are almost completely white [17], while
Edn3 gene mutations (lethal spotting) also result in severe melanocyte defects
[18]. Hirschsprung’s disease and Waardenburg syndrome are human homologues
[19]. The Ednrb gene is expressed in a spatially and temporally- controlled
manner during embryonic development, suggesting that Ednrb signaling is
required at several points during melanocyte development but not apparently for
postmigratory epidermal proliferation, differentiation, and survival [20].
Moreover, endothelin-3 induces the preferential expansion of melanocytes in
undifferentiated neural crest cell cultures [16].
Melanocyte precursors interact with dramatically varying micro-environments

during their highly regulated migration to the epidermis and hair follicle. This
migration will be facilitated/modulated by the expression on the migration
substratum of both integrins [21] and extracellular matrix molecules [22]. Indeed,
abnormal levels of extracellular matrix components on the migration substratum
occur in many mutants exhibiting abnormalities of neural crest migration [22].
The expression of cadherins also changes dramatically along the path of
migrating melanoblasts/melanocytes, with E-cadherin expression being restricted
to the epidermis while P-cadherin expression is observed in the hair matrix [23].
Thus, a complex orchestration of multiple signaling events, both permissive and
nonpermissive, influence the migratory pathway taken by melanocytes en route
to the hair follicle during development (Fig. 1).
After melanocytes have reached the human epidermis via the dermis (7 weeks
EGA), some of them enter the forming pilosebaceous units 2 weeks later [7].
There, they distribute randomly as cells positive and negative for
dihydroxyphenylalanine (DOPA) oxidase activity [24]. Melanogenic melanocytes
are found throughout hair follicle development from the hair germ stage onwards
[25]. When the hair fiber formation commences, melanocytes concentrate near
the basal lamina around the apex of the dermal/follicular papilla and, as
amelanotic cells, in the other root sheath. While melanocyte mitosis is observed
in the developing human epidermis (~14 weeks EGA), pigment cell mitosis is
rare in the hair follicle. This suggests that seeding of pigment cells from the
epidermis is the source of follicular melanocytes at this stage in development.
3
DISTRIBUTION, STRUCTURE, AND
MICROENVIRONMENT OF MELANOCYTES IN THE
GROWING PIGMENTED HAIR FOLLICLE
Our knowledge of the cell biology of the hair follicle pigmentary apparatus owes
much to the classic descriptions of Chase, Fitzpatrick, Burnet, Kukita, Staricco,
Billingham and Silvers, Straile, Mottaz, and Zelickson [26]. Recent availability
of melanocyte-specific antibodies and sophisticated molecular biology
techniques has added further refinements. The growing anagen VI hair follicle is
a useful starting position for a description of the cell biology of the hair
pigmentary unit and can be divided into four compartments on the basis of
DOPA, Masson silver, toluidine blue, and thionine staining patterns for
melanocytes. DOPA-positive melanotic melanocytes occur in only two
locations, the outer root sheath of the infundibulum and around the upper
follicular papilla. DOPA-negative amelanotic melanocytes are distributed in the
mid-to-lower outer root sheath and also in the peripheral and most proximal hair
bulb (Fig. 1). Some minor variations on this theme occur, however, including
rare outer root sheath distribution of melanogenically active melanocytes in the
scalp hair bulbs of blacks [27] and Chinese [28] people, among others, and in
300 TOBIN
FIGURE 1 Melanocyte development. Melanocyte migrate from the neutral crest to the skin
and hair follicle along stereotyped routes. Changes in cell differentiation status occur as
they progress along these pathways. MITF, microphthalmia protein; PDGF(R), platelet-
derived growth factor receptor; ET-3, endothelin 3; EDNRB, endothelin B receptor;
bFGF, basic fibroblast growth factor; SCF, stem cell factor; KIT, stem cell factor
receptor; ECM, extracellular matrix. (Adapted from Alhaidari Z et al. Vet Dermatol 1999:
10, 3–16.)
some cases of acute alopecia areata [29]. Positive morphological identification of

amelanotic melanocytes is usually achieved by means of electron microscopy,
which relies on the presence of premelanosomes. In the absence of these
organelles, the unequivocal identification of immature melanocytes can be

difficult.
Our identification of melanocytes has been greatly assisted by the availability
of melanocyte-lineage-specific antibodies, though these usually detect various
differentiation markers that may not yet be expressed on earliest melanoblast/
melanocyte precursors. The monoclonal antibody NKI/beteb detects a
premelanosome glycoprotein gp100 [30], as well as all DOPA-positive cells and
melanocytes producing premelanosomes [31, 32]. In our hands this antibody
does not, however, detect fully amelanotic melanocytes (e.g., melanocytes
located in the mid to lower outer root sheath of Caucasian skin types I/II).
Similarly, very heavily pigmented melanocytes in vitro show reduced NKI/beteb
expression, perhaps reflecting decreased melanosome organellogenesis in these
cells (Tobin et al., unpublished data). While DOPA-oxidase activity of tyrosinase
may not be detectable in amelanotic hair follicle melanocytes, the protein itself
may be detected in some of these cells [33]. Moreover, amelanotic melanocytes
may also express ckit and bcl-2 [34], although these cells do not also express the
melanogenic enzymes tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2)
[31].
The hair bulb is the only site of pigment production for the hair shaft, and it
contains both highly melanogenic melanocytes [35] and a minor subpopulation
of poorly differentiated bipolar amelanotic melanocytes [32] (Figs 1 and 2). The
role of amelanotic melanocytes in hair pigmentation is unclear (see later), though
it has been speculated that these cells represent a pool of “transient” melanocytes
that migrate from precursor melanocyte stores in the upper outer root sheath [31, 32,
36–38], (Fig. 2). Melanogenically active melanocytes are restricted to the upper
hair matrix of the anagen hair follicle, just below the precortical keratinocyte
population. This location correlates with the transfer of melanin during anagen
to the hair shaft cortex, less so to the medulla, and, very rarely, the hair cuticle.
In a manner akin to the epidermal-melanin unit of the skin, melanogenically-
active melanocytes in the hair bulb form functional units with neighboring
immature precortical keratinocytes. The follicular melanin unit resides in the
proximal anagen bulb, an immunologically distinct region of the skin [39]. This
so-called immune-privileged site [39] consists of 1 melanocyte to 5 keratinocytes
in the hair bulb as a whole and 1:1 in the basal layer of the hair bulb next to the
follicular papilla. Melanogenic bulbar melanocytes interact closely with the
follicular papilla, including via the thin/permeable basal lamina that separates
them from the mesenchymal follicular papilla. By contrast, each epidermal
melanocyte is associated with 36 “viable” keratinocytes in the fully
immunocompetent epidermalmelaninunit [40].
While follicular melanocytes are derived from epidermal melanocytes during
hair follicle morphogenesis, these pigment cell subpopulations diverge in many
important ways as they distribute to their respective distinct compartments
(Table 1). For example, hair bulb melanogenic melanocytes are larger, have
longer and more extensive dendrites, contain more developed Golgi and rough
302 TOBIN
FIGURE 2 Human scalp hair bulb melanocyte primary culture. This culture contains a
mixture of highly differentiated pigmented and dendritic bulbar melanocytes (M-Mc) and
poorly differentiated bipolar amelanotic melanocytes (Pre-Mc).
endoplasmic reticulum, and produce melanosomes two to four times larger than
those in epidermal melanocytes [41] (Figs 3 and 4). While melanin degrades
almost completely in the differentiating layers of the epidermis, eumelanin
granules transferred into hair cortical keratinocytes remain minimally digested
[41]. In this way, a eumelanic Caucasian individual may have black hair but very
fair, freckle-free, skin (e.g., blue-eyed, “black” Irish). By far the most striking
difference between these two melanocyte subpopulations, and one with
significant implications for the regulation of hair pigmentation, is the observation
that the activity of the hair bulb melanocyte is under cyclical control and that
melanogenesis is tightly coupled to the hair growth cycle [42]. Epidermal
melanogenesis, by contrast, appears to be continuous [43] (Table 1).
Much current research is focused on the mechanism (and regulation) of
melanin granule transfer from the melanocyte to the keratinocyte. Transfer of
melanin to cortical cells of the growing hair shaft is presumed to be similar to
that in the epidermis [44, 45]. It is widely accepted that this occurs via
cytophagy, where the keratinocyte, as active partner, phagocytoses the tips of
melanocyte dendrites that contain mature stage IV melanosomes [46]. Whatever
the exact mechanism(s), it is likely that melanocyte dendricity is critical in
melanin transfer, since the dendrites of melanotic bulbar melanocytes in dilute (d)
and pink-eye dilute (p) mouse mutants are abnormally short. Notably, myosin V
(encoded by the dilute gene) has also been proposed as a molecular motor
involved in dendrite outgrowth in mammalian melanocytes [47] and mutations at
this locus are associated with dilution of hair color [48].
TABLE 1 Differences Between Hair Follicle Melanocytes and Epidermal Melanocytes in Adult Human Skin
BIOLOGY OF HAIR PIGMENTATION
303
304 TOBIN
aTRH, thyrotropin releasing hormone; MSH, melanocyte-stimulating hormone; ASP, agouti signaling protein; ACTH, adrenocorticotroph-
hormone; DHT, dihydrotestosterone; MHC, major histocompatibility complex.
Source: From Ref. 41.
FIGURE 3 Transmission electron micrograph of a hair bulb melanocyte in a human scalp

anagen hair follicle. Note large numbers of maturing and fully mature melanosomes.
4
MELANOGENESIS
4.1
Melanosome Biogenesis
The process of melanogenesis can be divided into the formation of the
melanosome—the morphologically and functionally unique organelle in which
melanogenesis occurs—and the biochemical pathway that converts L-tyrosine
into melanin. Both processes are under complex genetic control that encodes a
range of enzymes, structural proteins, transcription factors, receptors, and growth
factors. While there is no evidence that melanosome biogenesis is significantly
different in follicular and epidermal melanocytes, we must keep an open mind.
Melanosome structure correlates with the type of melanin produced.
Melanocytes in black hair follicles contain the largest number of, and most
electron-dense, melanosomes (eumelanosomes), each with a fibrillar matrix [26]
(Fig. 4). Brown hair bulb melanocytes contain eumelanosomes that are
somewhat smaller, but phenotypically similar, while blonde hair bulbs produce
poorly melanized melanosomes with often only the melanosomal matrix visible.
Red hair pheomelanosomes contain a vesicular matrix but with melanin
deposited irregularly as blotches. Notably, albino hair melanosomes contain
normal-appearing early premelanosomes that fail to melanize. Both
306 TOBIN
FIGURE 4 Transmission electron micrograph of a part of a hair bulb melanocyte in a

human scalp anagen hair follicle. Note the presence of early premelanosomes (I and II),
maturing melanosomes (III), and fully mature ellipsoidal eumelanosomes (IV).
eumelanogenic and pheomelanogenic melanosomes can exist in the same normal

human melanocyte [49].
The formation and maturation of eumelanosomes is a subject of intense
research. Two views have emerged. The first indicates that the enzymatic
elements required for melanogenesis are delivered via coated vesicles to
melanosomes that originate from the endoplasmic reticulum and Golgi apparatus
[50]. An alternate interpretation of melanosome biogenesis, based on the
purification and analysis of early melanosomes, suggests that tyrosinase is sorted
to early endosomes by the adaptor protein-3 system (from the trans-Golgi
network) and from there to late endosomes. These then fuse with stage I
melanosomes [51]. While stage I melanosomes contain tyrosinase and other
melanogenesis proteins, these are believed to remain catalytically inactive until
subsequent protein cleavage events release them into the melanosome interior.
These events are associated with a change in melanosome shape, from spherical
to ellipsoidal, and the formation of an intramelanosomal fibrillar network
(eumelanosomes). Melanogenesis commences when tyrosinase and other relevant
enzymes are cleaved and is dependent on an acidic environment that is provided
by proton pumps. While tyrosinase is the rate-limiting enzyme of melanogenesis,
several other proteins need to be recruited and transported to the melanosome for
full functioning of these melanocyte-specific organelles. These include
tyrosinase-related protein 2 (TRP-2)—dopachrome tautomerase (DCT), gp100, p-
protein, and some of the members of the lysosome-associated membrane protein
family (LAMP-1,-2 and-3). It is still unclear just how and when these substances
are incorporated into the melanosome, though members of the adaptor protein
system (e.g., AP-3), and protein folding-assembly proteins (e.g., calnexin) and
small GTP binding proteins (e.g., Rab 5, 7) are likely to be involved.
Less is known about the events involved in the formation of the
pheomelanosome that produces the red/yellow melanin. In contrast to the
organized fibrillar network that characterizes eumelanosomes, pheomelanosomes
contain a vesiculoglobular matrix apparently derived from the fusion of
vesiculoglobular bodies with the stage I melanosomes. Tyrosinase activity
appears earlier in these melanosomes, such that pheomelanin is already deposited
in stage II melanosomes [50].
4.2
Formation of Melanin
The biochemical reaction that converts the amino acid L-phenylalanine, via L-
tyrosine, into a complex and heterogeneous group of compounds called melanins
can be broadly divided into the following steps:
1. The hydroxylation of L-phenylalanine/L-tyrosine to L-DOPA, the limiting

step in melanogenesis
2. The dehydrogenation of L-DOPA—the precursor for both (1) eumelanins
and pheomelanins and (2) the catecholamines
3. The dehydrogenation of dihydroxyindole (DHI) to yield melanin pigment
Of these steps, both eumelanogenesis and pheomelanogenesis require the

oxidation of DOPA to dopaquinone. Thereafter, the conversion of dopaquinone
to leukodopachrome signals eumelanin production, while the addition of cysteine
to dopaquinone to yield cysteinyldopa occurs in pheomelanin production (Fig. 5).
The constitutive color of a person’s skin and hair is due to absolute tyrosinase
activities rather than protein expression. Thus, tyrosinase regulation is critical,
being controlled not only by the supply of L-tyrosine but also by the stability/
activity of tyrosinase and tyrosinase-related proteins. Both L-phenylalanine and L-
tyrosine access the melanocyte, the former via the neutral amino acid Na+/Ca2+
ATPase anti-porter system and the latter by facilitated diffusion. L-phenylalanine
is converted to L-tyrosine via phenylalanine dehydroxylase (PAH) activity,
activities of which correlate positively with skin phototypes. This turnover from
L-phenylalanine to L-tyrosine is regulated by the rate-limiting cofactor (6R) -L-
erythro-5, 6, 7, 8-tetrahydrobiopterin (6BH4). Although 6BH4 can also inhibit
tyrosinase allosterically, this affects only the hydroxylation of L-tyrosine to L-
DOPA and not also the oxidation of L-DOPA to L-dopaquinone. The 6BH4-
tyrosinase inhibitor complex can be activated by UVB photooxidation or
independent of light by complexing with -MSH [52].
308 TOBIN
FIGURE 5 Biosynthetic pathway of melanin. (From Ref. 52.)

For eumelanogenesis, L-DOPA needs to be oxidized by tyrosinase to L-

dopaquinone and again by tyrosinase from DHI to indole-5, 6-quinone. Thus,
tyrosinase with its tyrosinase hydroxylase and DOPA oxidase activities not only
initiates the melanogenesis pathway but rapidly advances it. Both tyrosinase-
related proteins [TRP-1 and DCT (TRP-2)] are important for maintaining the
stability of tyrosinase at the melanosomal membrane. Eumelanogenesis is
critically dependent on the velocity of the tyrosinase reaction, although it is also
stimulated by TRPl and DCT. For pheomelanogenesis, the formation of
cysteinyldopa is required [53]. Cysteinyldopa is further oxidized in multiple
complex steps that may involve tyrosinasedependent, tyrosinase-independent
reactions as well as glutathione reductase and peroxidase activities to form
pheomelanin.
5
THE REGULATION OF HAIR FOLLICLE
MELANOGENESIS
The pigmentation of hair fibers is affected by numerous intrinsic factors
including hair-cycle-dependent changes, body distribution, racial and gender
differences, variable hormone responsiveness, genetic defects, and age-
associated change. Study of hair pigmentation may even be complicated by the
effects of extrinsic variables including climate and season, infestations,
pollutants, toxins, and chemical exposure. Given that melanosome biogenesis
and melanogenesis involve multiple steps, it is perhaps not surprising that
positive and negative regulators of hair follicle melanogenesis will involve
multiple biological factors. These include growth factors, cytokines, hormones,
neuropeptides and neurotransmitters, eicosanoids, cyclic nucleotides, nutrient
microelements, and cations and anions [43]. These may act via autocrine,
paracrine, and endocrine mechanisms. While much of the literature pertains to
epidermal melanocytes, it is likely that similar mechanisms (with the notable
possible exception of UVR-induced changes) will also operate in follicular
melanogenesis. These melanogenesis regulators can be divided into positive and
negative regulators. Examples of positive regulators of melanogenesis are: the
proopiomelanocortin (POMC) peptides -MSH, ACTH, and -endorphin,
endothelins 1 and 3, and prostaglandin E, while negative regulators include
melanin itself, interleukins and 6 (IL-1, IL-6), tumor necrosis factor (TNF- ),
and transforming growth factor (TGF- ) [54].
5.1
Regulation of Hair Pigmentation by Endocrine, Paracrine,
and Autocrine Hormones
Because little is known specifically about the regulation of human hair
pigmentation, we rely heavily on studies of the regulation of pigmentation in the
310 TOBIN
rodent coat. UVB radiation does not penetrate to the melanogenic cells of the
anagen hair bulb located in the subcutaneous fat and so is unlikely to influence
the follicular melanin unit directly. Studies in guinea pigs have shown that -
MSH increased the proportion of black to gray hairs when administered
intramuscularly [55]. However, the injection of both -MSH and the potent
synthetic analogue [Nle4, D-Phe7]- -MSH into human skin results in increased
melanogenesis, particularly of sun-exposed skin [56]. No effect however, was
seen in hair follicles. In support of this, we have recently found that the
expression of -MSH is very low to undetectable in pigmented hair bulb
melanocytes versus epidermal counterparts both in vivo and in vitro (Kauser and
Tobin, unpublished data). However, it is likely that the MC1 receptor (MCR-1),
the product of the extension (e) locus and cognate receptor of -MSH, is an
important positive regulator of hair pigmentation [54]. This G-protein-coupled
membrane receptor is activated upon binding of POMC-derived ACTH, -MSH,
and -MSH peptides. The resultant signal transduction cascade results in the
activation of adenylate cyclase activity and subsequent cAMP production
followed by increased melanocyte proliferation, melanogenesis, and dendrite
formation. Agouti signaling protein (ASP) is likely to be an important negative
regulator of hair pigmentation because it competitively inhibits the binding of -
MSH to MCR-1. In this way ASP not only switches melanin synthesis from
eumelanogenesis to pheomelanogenesis but also inhibits melanogenesis overall.
It is notably that the expression and translation of POMC gene products for
MCR-1 are expressed in the skin in a hair-cycle-dependent manner; being low at
telogen and high during anagen development [57]. Moreover, this accumulation
of POMC products is found predominantly in the outer root sheath follicular
keratinocytes in the scalp less in the overlying epidermis, suggesting that the
activity of local POMC/MCR-1 axis plays an important role in the physiological
regulation of anagen-associated hair pigmentation. In this context, it is important
to note that polymorphism in the MCR-1 gene has been strongly linked to red
hair and fair skin in humans [58]. Importantly, -MSH can remove the cofactor
6BH4 from the 6BH4-tyrosinase inhibitor complex, suggesting that the relative
amounts of both 6BH4 and -MSH may be involved in the regulation of
pigmentation levels in the melanocyte [59].
Melanocytes in the epidermis expressbcl-2, an antiapoptotic oncogene
associated with cell survival. Follicular melanocytes, especially those located in
the outer root sheath, also express this survival factor. It has been proposed that
bcl-2 inhibits cell death particularly in areas where reactive oxygen species are
generated (e.g., melanogenesis) by regulating antioxidant pathways [60].
Similarly, hepatocyte growth factor (HGF) may regulate melanocyte survival,
proliferation, and differentiation in vivo in a paracrine manner and even alter
melanocyte distribution within the skin via downregulating E-cadherin
expression on melanocytes [61].
6
MODULATION OF THE HAIR PIGMENTARY UNIT
DURING THE HAIR GROWTH CYCLE
Active pigmentation occurs only during the hair growth phase (anagen), which in
human scalp hair can be very long (up to 10 years) (see later: Figs. 6c and 8c).
The extended anagen of human scalp hair, together with its mosaic pattern of
hair growth, hinders systematic analysis of melanocyte dynamics during the
human hair cycle. By contrast, the short growth phase (15–17 days),
synchronous hair growth pattern, restriction of melanogenically active truncal
melanocytes to the hair follicles [62], and the linkage of murine melanogenesis
with anagen [42] all make the C57BL/6 mouse an invaluable model for human
hair pigmentation investigation. Moreover, the intense pigmentation of C57BL/6
hair facilitates easy staging of anagen (gray to black skin) and telogen (pink
skin) [63].
6.1
Telogen-to-Anagen Transition
The relatively quiescent telogen hair germ contains all cell precursors needed to
reconstitute a fully developed anagen VI hair follicle [64] (Figs. 6a, 7a, 8a).
Telogen C57BL/6 skin does not contain tyrosinase (mRNA/ protein), TRP-l
protein, or melanin, although very low level activity for tyrosine hydroxylase
may be detected [65] (Fig. 7b). During the first day or two of anagen induction,
tyrosinase messenger protein becomes barely detectable, a finding that fits with
the presence of occasional amelanotic melanocytes containing tyrosinase-
positive premelanosomes. At this stage, the follicular papilla pools high
concentrations of L-phenylalanine, a potential requirement for the supply of L-
tyrosine for melanogenesis [52] (Fig. 7c). Thus, in earliest anagen the levels of
6BH4, GTP-cyclohydrolase 1, and phenylalanine hydroxylase (PAH) are high.
These conditions support the production of high amounts of L-tyrosine from L-
phenylalanine—a prerequisite for melanogenesis. Activities of all three drop
significantly by anagen III and remain low until the next telogen (Fig. 7c). Low
levels of 6BH4 are necessary during pigment production to prevent the allosteric
inhibition of tyrosinase. Just prior to this early anagen-associated drop (i.e.,
anagen II), tyrosinase message, protein, and activity all begin to increase rapidly
to peak at early anagen VI (full anagen) (Fig. 7a-b).
312 TOBIN
FIGURE 6 Schematic representation of the hair follicular pigmentary unit during the hair growth cycle, (a) Telogen: note the presence of
amelanotic melanocytes in the outer root sheath (ORS) of the telogen club and residual melanin in the dermal papilla (DP), (b) Early anagen:
note the reconstitution of the hair pigmentary unit, including dendritic melanocytes, in the developing anagen hair bulb, (c) Full anagen VI:
note the melanin is transferred actively into the hair shaft at this stage of the hair cycle, (d) Early catagen: note that the resorption of the proximal
hair follicle is associated with the loss of some highly differentiated hair bulb melanocytes by apoptosis. IFD Mc, infundibular melanocytes;
Epi Mc, epidermal melanocytes.
FIGURE 7 Hair-cycle-associated change in melanogenesis-associated proteins and

enzymes in the C57BL/mouse model. (a) Western blot analysis of the expression of
tyrosinase protein (TP) and analysis of DOPA oxidase (DO) activity after incubation of
skin extract with L-DOPA, during the hair cycle. (From Ref. 37.) (b) Changes in tyrosine
hydroxylase activity of tyrosinase during the hair cycle (From Ref. 37.) (c) Changes in L-
phenylalanine hydroxylase activity during the hair cycle (From Ref. 59.) (d) Changes in
dopachrome tautomerase (DCT/TRP-2) activity during the hair cycle. (From Ref. 37.)
The anagen-associated stimulation of undifferentiated melanocytes/

melanoblasts located in the telogen secondary germ predates the melanogenic
314 TOBIN
stimulus delivered during anagen III (Figs. 6b, 7a-b). This is followed by active
melanogenesis and subsequent transfer of mature melanosomes into
keratinocytes of the precortical matrix. Combined DOPA-reaction cytochemistry
and electron microscopy reveal rare DOPA-positive Golgi complexes in some of
these immature melanocytes, thereby providing evidence of low-level tyrosinase
activity [53]. Melanocytes in the S phase of the cell cycle have been reported as
early as anagen II [66], and significant proliferation is clearly apparent in anagen
III [67]. Mitosis is also observed in melanogenically active cells, indicating that
melanocyte differentiation does not preclude mitotic activity. Bulbar
melanocytes during the transition from anagen III to anagen VI increase in their
dendricity, develop more Golgi and rough endoplasmic reticulum, increase the
size and number of their melanosomes [68], and begin to transfer mature
melanosomes to precortical keratinocytes (Figs. 6a-6c, 8b,c).
6.2
Anagen-to-Catagen Transition
Even before catagen-associated structural changes are apparent in the hair bulb,
the earliest signs of imminent hair follicle regression include the retraction of
melanocyte dendrites and the attenuation of melanogenesis during late anagen VI
[38] (Figs. 6d, 8a-c). Limited keratinocyte proliferation continues for a while, so
the most proximal telogen hair shaft remains unpigmented. The functional
relevance of this keratinocyte-melanocyte asynchronicity is unclear, although the
switch-off of melanogenesis and reduction in dendricity would restrict the
transfer of melanin to precortical keratinocytes. One can detect already a
dramatic and rapid drop in levels of active tyrosinase beginning during late
anagen VI itself (Fig. 7a, b, d). There are moderate reductions in TRP-2 (DCT)
and DHICA-CF (dihydroxyindole carboxylic acid-conversion factor) activity
from mid to late anagen VI, and activities are lowest during catagen (Fig. 7d).
The termination of melanogenesis may reflect a swamping of a melanogenesis-
dependent signaling system or the induction of melanogenesis inhibitory factors
(e.g., IL-1, IL-6, IFN- , TGF- , TNF- , or corticosteroids) [43]. Alternatively,
the low supply of L-tyrosine in the now reducing hair bulb environment inhibits
melanogenesis, until increased production of GTP-CH-1, 6BH4, and PAH occurs
again during telogen in preparation for optimal melanogenesis conditions during
the subsequent anagen phase.
6.3
Fate of Pigmented Melanocytes During Catagen
A long-enduring enigma of both hair follicle and pigment biology concerns the
fate of the hair bulb melanocytes during catagen (Figs. 6d, 9a–c). Particularly
relevant questions include these: where do these melanocytes go during catagen
and telogen, and where do they originate from when follicular melanogenesis is
FIGURE 8 Localization of melanocytes during the telogen-to-anagen transformation. (a)

NKI/beteb-positive melanocytes (arrows) in the telogen human scalp hair follicle. (b)
NKI/beteb-positive melanocytes (arrows) in the early anagen human scalp hair follicle.
(c) Full reconstruction of the hair follicle pigmentary unit in anagen VI human scalp hair
follicle. DP, dermal papilla.
resumed during the next anagen phase. Melanogenically active melanocytes are
no longer detectable in the proximal hair follicle during catagen. However, their
“disappearance” is not unheralded, and residual melanin generated during anagen
can be seen “deposited” in the follicular papilla [38] (Fig. 9a–b). A long-held
view in hair biology is that the hair bulb melanocyte system is a self-perpetuating
arrangement, whereby melanocytes involved in the pigmentation of one hair
generation are also involved in the pigmentation of the next [69]. This view
relies on an interpretation of melanocyte dedifferentiation (e.g., retraction of
dendrites and reduction of melanogenesis) (Fig. 9b) and redifferentiation during
the subsequent anagen phase. While there is evidence of some plasticity in the
hair follicle pigmentary unit (Fig. 9b), the level invoked by the self-perpetuating
theory would imply a degree of plasticity not seen in most nonmalignant cell
systems. Moreover, fully differentiated bulbar melanocytes would also need to
survive/avoid the extensive apoptosis-driven regression of the hair bulb [70, 71]
by actively suppressing apoptosis.
Thus, our current view suggests that many of the so-called redifferentiating
melanocytes in early anagen correspond to newly recruited immature
melanocytes derived from a melanocyte reservoir [38, 72] and are not reactivated
from preexisting hair bulb melanocytes that were melanogenically active during
316 TOBIN
the preceding anagen phase. This is supported by the observation of a population

of immature DCT+ melanocytes, not affected by blocking anti-ckit antibody, in
the murine bulge [73]. It is possible, however, that some new generation
melanogenically-active melanocytes derive from a population of catagen-
surviving, post-melanogenically active cells. Indeed, low numbers of apparently
dendritic melanocytes can be detected in the retreating epithelial strand of
catagen hair follicles undergoing active resorption via apoptosis [74], Tobin et
al., unpublished data] (Fig. 9c). However, these weakly melanogenic or
nonmelanogenic cells lack tyrosinase and TRP-1 expression and may in fact
represent the poorly differentiated melanocytes that codistribute with pigmented
bulbar melanocytes in anagen hair matrix [35].
We recently reported, that some highly melanotic (terminally differentiated?)
hair bulb melanocytes the C57BL/6 mouse model do not survive catagen [75].
Deletion of individual melanotic melanocytes by apoptosis was confirmed by
using well-described ultrastructural features (Fig. 9d, e) and TUNEL/TRP-1
colocalization. That some, if not most, highly pigmented hair bulb melanocytes are
indeed lost during catagen is further supported by the observation that the vast
majority of cells attached to the basal lamina of the catagen hair bulb are
epithelial.
6.4
Catagen-Associated Pigment Incontinence
Not all the pigment formed during anagen-associated melanogenesis is
incorporated into the hair shaft. Indeed, “excess” pigment appears to be removed
from the hair follicle. It is likely that this is related to melanocyte apoptosis and
that resultant pigment-containing apoptotic fragments enter the follicular papilla
(Fig. 9b), epithelial strand, or connective tissue sheath (CTS) of catagen
(Fig. 9a). Pigment incontinence may also be detected in the epithelial sac of the
telogen hair follicle. The precise mechanism(s) of this pigment redistribution is
unclear, although it is likely to involve phagocytosis, particularly by
macrophages and Langerhans cells (Fig. 9f–g), which increase in numbers during
hair follicle regression [39], or by follicular papilla fibroblasts themselves. Rare
macrophages may even be detected in the human hair bulb matrix during late
anagen, where they may ingest melanin, derived presumably from degenerating
hair bulb melanocytes [75, 76]. Moreover, Langerhans cells are also more
commonly detected in the regressing human catagen hair bulb [77] (Fig. 9f-g), in
canities [28], and in alopecia areata [78]. Langerhans cells may remove pigment
from the regressing hair matrix to the follicular papilla via direct phagocytosis or
via Langerhans granule-associated endocytosis of (pre) melanosomes (Fig. 9f–
g). Melanin removal may also occur after uptake by a small number of outer root
sheath keratinocytes [77].
FIGURE 9 Changes in the hair follicle pigmentary unit during hair follicle regression
(catagen). (a–b) NKI/beteb-positive melanocytes (arrows) in the late catagen human scalp
hair follicle. Note melanin debris (Me) in the epithelial strand (ES) of the catagen hair
follicle. Melanin debris is also located in the dermal papilla (DP). (c) NKI/beteb-positive
melanocytes (arrows) in very late catagen human scalp hair follicle. Note that some
melanocytes located in the epithelial strand exhibit a dendritic phenotype. (d–e)
Transmission electron micrographs showing melanocyte apoptosis (AP) in murine pelage
early catagen hair follicle. Note the rounding up and condensation of affected
melanocytes, and that these contain melanocyte-specific premelanosomes (PM). DP,
dermal papilla. (From Ref. 75.). (f) Transmission electron micrograph showing a portion
of a Langerhans cell transferring melanin granules from the matrix (MX) to the dermal
papilla (DP). Note the characteristic Langerhans cell granules (Lg) (From Ref. 77.) (g)
Transmission electron micrograph showing a portion- of a Langerhans cell containing
melanin granules enclosed within forming and expanded Langerhans granules (arrows)
(From Ref. 27.)
318 TOBIN
7
AGE-RELATED ALTERATIONS IN THE HAIR
PIGMENTARY UNIT
The hair pigmentary unit goes through significant age-associated changes that
range from short, fine, and usually unpigmented lanugo hair at about 3 months
of intrauterine life to the white hair of old age. In between is childhood
intermediate hair that after puberty may become, as with scalp hair, more deeply
pigmented terminal hairs. A partial reverse sequence is seen with the reduction in
pigmentation in miniaturizing hair follicles in androgenetic alopecia. However, it
is in canities where one observes the most dramatic age-related change in hair
pigment.
7.1
Molecular Aspects of Melanocyte Aging
For every decade after 30 years of age the number of pigment-producing
melanocytes in exposed/unexposed epidermis decreases by 10 to 20% [79]. This
age-associated loss of DOPA-positive epidermal melanocytes occurs all over the
body and is associated with a very gradual reduction in skin color. By contrast,
age-linked loss of color from hair is dramatic, suggesting that the hair
pigmentary unit has a different “melanogenetic clock.” It has been observed that
loss of melanocyte replicative potential in vitro is associated with increased
melanin content. This is particularly so after long-term continuous exposure to
cAMP inducers (e.g., cholera toxin), which do not engage the melanocortin-1
receptor [80]. Similarly, millimolar concentrations of L-tyrosine (melanin
precursor) abrogate proliferation in cultured “presenescent” pigmented
melanocytes, with proliferation continuing only in amelanotic cells [81]. On
reaching senescence, melanocytes express increased levels of cyclin-dependent
kinase (CDK) inhibitors (e.g., p21 and p16). Accumulation of oxidative damage
is an important determinant in the rate of cell aging, although it is unclear
whether it is the primary cause of aging. Reactive oxygen species (ROS) damage-
DNA (both nuclear and mitochondrial), which leads to the accumulation of
mutations, induces oxidative stress, and also induces antioxidant mechanisms. It
is possible that this antioxidant system becomes impaired with age, leading to
uncontrolled damage to the melanocyte itself from its own melanogenesis-related
oxidative stress.
Melanin synthesis, by its very nature, produces mutagenic intermediates [82],
and thus the induction of replicative senescence in melanocytes is likely to be an
important protective mechanism against cell transformation [83]. The
extraordinary melanogenic activity of pigmented bulbar melanocytes, which may
continue for up to 10 years in some scalp hair follicles, is likely to generate large
amounts of ROS via the oxidation of tyrosine and DOPA to melanin [84]. If not
adequately removed, an accumulation of these ROS will generate significant

oxidative stress both in the melanocyte itself and also in the highly proliferative
anagen hair bulb epithelium. Thus, in these circumstances, melanogenic bulbar
melanocytes are perhaps best suited to assume a postmitotic, terminally
differentiated “(Pre) senescence” status to prevent cell transformation.
7.2
Onset and Progress of Canities
A characteristic feature of bulbar melanocytes is their extremely high melanin
load throughout the entire time the pigmented hair fiber is forming during anagen
—up to 10 years in the human scalp. This represents a phenomenal synthetic
capacity for melanin production (Figs. 3, 4, 6c and 8), whereby a relatively small
number of melanocytes can, in a single hair growth cycle, produce sufficient
melanin to intensely pigment up to 1.5 m of hair shaft. Moreover, they do this
within the context of a melanin-laden cytoplasm, unlike melanogenically active
epidermal melanocytes that retain few fully mature melanosomes in their
cytoplasm at any one time. This “melanin loading” of bulbar melanocytes is
likely to make these cells much more vulnerable than epidermal melanocytes to
the toxic elements of melanogenesis.
The synthetic capacity of bulbar melanocytes is greatest during youth when
the scalp follicular melanin unit is only a few cycles old. On average, therefore,
an individual scalp hair follicle will experience approximately 7 to 15
melanocyte seedings/replacements from the presumptive reservoir in the outer
root sheath to the hair bulb in the average “gray-free” life span of 45 years [85].
Interestingly, repeated plucking of hair from vibrissae follicles leads to the
eventual regrowth of gray hair [86], though the associated tissue injury
complicates the interpretation of this finding. Furthermore, the precise rate of
canities progression is also complicated by the observation that with advancing
age hair follicles remain longer in telogen, suggesting that epithelial stem
activation/migration may also become more sluggish with age.
The onset and progression of hair graying correlates closely with chronological
aging and occurs to varying degrees in all individuals, regardless of gender or
race. Age of onset also appears to be hereditary, occurring usually in late fourth
decade [85]. The average age for Caucasians is mid-30s, for Asians, late-30s, and
for Africans mid-40s. Hair is said to gray prematurely if it occurs before the age
of 20 in whites, before 25 in Asians, and before 30 in Africans. The progress of
canities is entirely individual: a good rule of thumb is that by 50 years of age,
50% of people have 50% gray hair [85]. Clearly, the darker the hair, the more
noticeable early graying will be. However, graying can be more extensive in dark
hair before total whitening is apparent; the reverse is true for blond hair. Graying
first appears usually at the temples and spreads to the vertex and then to the
remainder of the scalp, affecting the occiput last. Beard and body hair is usually
320 TOBIN
affected later. Graying often follows a wave that spreads slowly from the crown
to the occiput.
7.3
Pathogenesis of Canities
Perhaps surprisingly, “gray” hair has been considered to be illusory, a mere
impression of grayness provided by an admixture of fully white and fully
pigmented hair. However, canities can indeed affect individual hair follicles with
either a gradual loss of pigment over time and over several cycles, a gradual loss
of pigment along the same hair shaft (i.e., within the anagen phase of a single
hair cycle), or the hair fiber may appear to grow in fully depigmented (Fig. 10a-c).
While few pigment granules are present in truly white hair shafts, melanin
granules can be readily detected within the precortex of gray hair follicles
(Fig. 10b-d). Pigment loss in graying hair follicles is due a marked reduction in
melanogenically active melanocytes in the hair. Compared with fully pigmented
hair follicles, bulb of gray anagen hair follicles [87] true gray hairs show a much
reduced, but detectable, DOPA reaction as an indicator of tyrosinase activity
(Fig. 11a, b), while white hair bulbs are broadly negative (Fig. 11c). However,
there appears also to be a specific defect of melanosome transfer in graying hair
follicles, in as much as keratinocytes may fail to contain any melanin granules
despite being in close proximity to melanocytes with a moderate number of
melanosomes (Tobin et al., unpublished observations). Further evidence of some
defect in melanocyte-keratinocyte interaction is provided by the observation of
significant melanin debris both in the graying hair bulb and sometimes also in
the surrounding dermis. This abnormality is due to defective melanosomal
transfer to the cortical keratinocytes and/or to melanin incontinence due to
melanocyte degeneration. The remaining hair bulb melanocytes in canities-
affected anagen hair follicles often appear hypertrophic, although this may
reflect a reduction in dendricity rather than an overall increase in cell volume [88].
Ultrastructural analysis of the human gray hair matrix reveals melanocytes
with highly variable levels of melanogenesis (Fig. 12) [89]. In gray/ white hair
bulbs, remaining melanocytes contain fewer and smaller melanosomes and fewer
supporting organelles (e.g., Golgi apparatus). Interestingly, the remaining
melanosomes may be packaged within autophagolysosomes, suggesting that
these melanosomes are defective, perhaps even leaking reactive melanin
metabolites. Autophagolysosomal degradation of melanosomes is usually
followed by the degeneration of the melanocyte itself [90, 91]. The involvement
of ROS in the histopathology of canities is suggested by the observation that
melanocytes in graying and white hair bulbs may be vacuolated, a common
cellular response to increased oxidative stress [92]. Degenerative change in
canities-affected hair bulbs may resemble apoptosis (Fig. 12) and is reminiscent
of melanocyte degeneration in acute alopecia areata, where pigmented hair
follicles are preferentially targeted by an aberrant immune response [29]. Loss of
FIGURE 10 Changes in the hair follicle pigmentary unit during hair graying (i.e.
canities) (a) Macroscopic view of normal human scalp tissue exhibiting hair shafts that
are pigmented (P), gray (G), and white (W). (b-c) Frozen section of normal human scalp
showing pigmented (P), gray (G) and white (W) hair bulbs. (d) High-power view of gray/
white hair bulb showing presence of some melanin granules (Me) asymmetricaly
distributed in the precortex (PC) and some melanin incontinence (Me) in the dermal
papilla (DP). (e) Surpa-DP region of a normal human scalp graying hair follicle showing
focus of keratinocytes exhibiting an altered differentiation status (Diff). (f) Keratogenous
zone of normal human graying scalp hair follicle showing medulla (MD) formation
alongside precortex (PC).
melanocytes from canitiesaffected hair bulbs can apparently occur very rapidly.
Evidence for this can be found in the pigment incontinence located in the
follicular papilla and/ or connective tissue sheath of hair follicles that lack any
morphological evidence of melanogenesis or melanocytes in their hair bulb. The
presence of pigment debris in an amelanotic hair follicle would appear to
indicate the recentness of events responsible for loss of previously melanogenic
melanocytes.
322 TOBIN
FIGURE 11 DOPA oxidase activity in plucked human hair follicles with different pigmentation levels, (a) Pigmented hair bulb showing
intensely DOPA oxidase-positive melanocytes (arrow), (b) Macroscopically white hair bulb still containing a solitary DOPA oxidase-positive
and dendritic melanocyte (arrow), (c) White hair bulb showing total lack of DOPA oxidase positivity.
FIGURE 12 Transmission electron micrograph of canities affected human hair bulb. Note
the presence of a degenerative melanocyte (APM) exhibiting marked condensation
alongside a morphologically normal-appearing hair bulb melanocyte (MC) and
keratinocyte (KC).
The loss of active melanocytes from the hair bulb of graying and white hair
follicles may be associated with a parallel increase there in dendritic cells
(including Langerhans cells) [89]. The relocation of these antigen-presenting
phagocytic cells from the upper hair follicle to the lower follicle may be in
response to degenerative change the melanocyte population.
7.4
Does Melanocyte Loss Change Hair Follicle Function?
Given the close interaction between melanin-transferring melanocytes and
precortical keratinocytes that form hair shafts and accept melanin, it is likely that
bulbar melanocytes influence keratinocyte behavior in several ways. Melanin
transfer appears to promote decreased keratinocyte cell turn-over and increased
keratinocyte terminal differentiation, perhaps by altering intercellular calcium
levels. White beard hair has been shown to grow at up to three times the rate of
adjacent pigmented hair [93]. In this way melanosomes donated to keratinocytes
may act as regulators that control their level of cell differentiation and even
metabolic status [94]. Melanocytes may also influence neighboring keratinocytes
via the production of various cytokines, growth factors, eicosanoids, adhesion
molecules, and extracellular matrix [94]. Similarly, the ability of melanins to
provide a buffer for calcium is likely to have implications for cell function, given
324 TOBIN
the critical second-messenger/cell signaling role for calcium in melanogenesis,

melanosome transfer, and epithelial cell differentiation [95]. The saturation
binding of transition metals (e.g., iron, copper) to melanin provides yet another
effective anti-oxidant defense mechanism for the melanin-receivingkeratinocyte.
Further clinical evidence of melanocyte-keratinocyte interactivity can be seen
in the anecdotal impressions that gray hair is coarser, wirier, and more
unmanageable than pigmented hair. Indeed, gray hair is often unable to hold a
permanent or temporary set and is more resistant to incorporating artificial color.
These observations suggest significant change to the underlying substructure of
the hair shaft, whereby aging hair follicles may reprogram their matrix
keratinocytes to increase production of medullary, rather than cortical,
keratinocytes. (Fig. 10e, f).
7.5
Can Canities Serve as a Marker for Disease?
Although the interpretation is very controversial, there is increasing evidence to
suggest that graying may be a marker for general health status. Cigarette
smoking has been linked with premature gray and even hair loss [96], although
this may rather reflect smoking-related pathology that increase aging of many
body systems including pigmentation, or apoptosis may have been induced by
smoke genotoxin. Moreover, it has been reported that individuals with premature
canities are more likely to develop osteopenia than individuals without canities
[97] and that people who grayed before their twenties had lower bone mineral
density than those who grayed later. Purported associations between early onset
of gray hair and cardiovascular disease [98] or studies showing graying of hair,
male baldness, and facial wrinkling as additional risk factors for myocardial
infarction are less clear.
7.6
Use of Artificial Hair Colorants to Treat Canities
In the absence of natural regimens to recover lost hair color, many individuals
turn to hair colorants [41]. Such products are used very successfully and safely
by millions of individuals worldwide. Some studies however, have raised the
possibility that long-term usage of permanent hair dyes (particularly black dyes)
may be associated with a very small increased risk of developing certain cancers.
However, the findings of these small, poorly controlled, studies remain highly
controversial and importantly, have not been confirmed in much larger,
adequately controlled, recent studies [99, 100]. A small number of users may
develop chemical and allergic reactions and these may result in dermatitis and
even hair loss [101]. It would appear prudent therefore, to evaluate ways of
improving strategies to restore hair color, for example, by improving further
further hair dye safety and by reconsideration of ways to block or reverse the
process of hair graying itself.
7.7
Is Canities Reversible?
Canities-associated pigment loss results from loss of melanocytes from the
melanogenic zone of the hair follicle. By contrast, senile white hair follicles
retain amelanotic melanocytes in the outer root sheath. These cells for the most
part remain not only DOPA-negative and but also negative for most melanocyte-
specific markers [31]. While the precise role of outer root sheath amelanotic
melanocytes in hair and skin biology is far from clear, these cells can be
recruited for repigmentation/repopulation of the epidermis if necessary (e.g., in
vitiligo) [102]. Their failure to contribute to the pigmentation of senile white hair
follicles may reflect the lack of a permissive environment for their migration to
the melanogenic zone during early anagen. Only melanocytes that have
successfully migrated to the hair bulb appear to be susceptible to local
pigmentation-inducing influences (e.g., anagen-induced secretion of factors
derived from follicular papilla-derived). The provision of some of these stimuli
in vitro can result in the induction of melanogenesis in the amelanotic cells of
senile white hair follicles (Tobin et al., unpublished data). This finding suggests
that these cells retain the melanogenic machinery intact and so could be induced
to become active again in more permissive in vivo microenvironments.
The deficit in canities-affected hair follicles is likely to be multifactorial.
Primary among these may be defective migratory stimuli, particularly during the
critical stages of the hair cycle when cell-cell and cellmatrix interactions are highly
active. Several factors could theoretically be administered to canities-affected
scalp. Basic fibroblast growth factor, leukotriene C4, and endothelin 1 are potent
chemotactic factors at least in the Boyden chamber type of in vitro study [103].
Along with another potent melanocyte migration factor, stem cell factor (SCF),
these molecules regulate the expression of integrins on the surface of several cell
types including melanocytes themselves. These growth factors are produced in
the skin by keratinocytes [basic fibroblast growth factor (bFGF), endothelin 1]
and by fibroblasts, including the all-important and optimally located follicular
papilla fibroblasts (SCF, leukotriene 4) [104, 105]. SCF and its receptor ckit
have been directly implicated not only in the migration (via chemokinesis) of
melanoblasts into the hair follicle but also in their survival and proliferation
[106]. Spontaneous scalp hair repigmentation has been reported after radiation
therapy for cancer [107] or after inflammatory events (e.g., erythrodermic
eczema and erosive candidiasis of the scalp) [108]. Here, it is most probable that
reversal of canities resulted from radiation/cytokine-induced activation of outer
root sheath melanocytes, which in turn raises the attractive possibility that these
melanocytes may be induced to migrate and differentiate to naturally repigment
graying hair follicles. Another clinical scenario that provides an insight into both
326 TOBIN
the pathomechanism of canities and possibilities for pigment recovery is the not
too uncommon partial spontaneous reversal of canities that occurs during the
early stages of canities. Here, melanogenesis in deactivated bulbar melanocytes
may restart during anagen VI of the same hair growth cycle [28]. Study of hair
follicles at this point in canities may provide several clues to help us identify the
subtle changes in the hair follicle’s two melanocyte subpopulations.
8
DISORDERS AFFECTING THE HAIR FOLLICLE
PIGMENTARY UNIT
Hair follicle pigmentation disorders may be acquired, endogenously induced
hypomelanoses including circumscribed poliosis (e.g., alopecia areata and
vitiligo), genetic hypomelanoses including albinism and circumscribed poliosis
(e.g., piebaldism, Waardenburg’s syndrome), and endocrineassociated hair
dilution (e.g., homocystinuria, and sometimes in phenylketonuria). While a
detailed discussion of the many human disorders that entail hair color defects is
beyond the scope of this chapter, it is nonetheless useful to consider the
involvement of the follicular melanin unit in the pathogenesis of a few common
dermatoses.
8.1
Alopecia Areata
Shah Jahan, Marie Antoinette, and St. Thomas More are just a few historic
figures afflicted by sudden loss of hair color that are reported to have occurred
during dramatic periods of their lives. While early graying has been related to
anxiety of a more chronic type,“turning white overnight” (canities subita) is
anecdotally associated with episodes of acute fear or grief [109]. While there
have been many wild speculations to explain this phenomenon (e.g., spontaneous
bleaching), acute alopecia areata is now considered to be the most likely
explanation. Here, the disease process preferentially targets pigmented hair, and
increasing data indicate that the melanogenic follicular melanocytes are a
principal target in alopecia areata. Not only do pigmented bulbar melanocytes
undergo degenerative change in acute alopecia areata [110, 110] (Fig. 13), but
patients also produce antibodies to cytoplasmic antigens expressed in cultured hair
follicle melanocytes [111], and have lesional T cells that are pathogenic after
exposure to melanocyte antigens (Gilhar et al., personal communication). It has
been hypothesized that in alopecia areata, melanogenesis-associated proteins
produced during antigen III/IV may trigger an antipigmented hair follicle
immune response as a result of anagen leakage [112].
FIGURE 13 Selective targeting of hair bulb melanocytes in an individual affected with

alopecia areata. (a) Note that the total loss of functional melanocytes from the anagen hair
bulb is associated with the total redistribution of melanin granules (Me) to the dermal
papilla (DP). Melanin is also located within melanophages (Mg) located in the connective
tissue sheath and dermis (From Ref. 110.) (b) Transmission electron micrograph of an
anagen scalp hair follicle bulb in an individual affected by alopecia areata, showing
several degenerating melanocytes (APM). DP, dermal papilla; KC, keratinocyte (From
Ref. 110.)
8.2
Vitiligo
Vitiligo, a depigmenting disorder of the epidermis, can occasionally result in
leukotrichia in the depigmented skin (leukoderma). While the status of hair bulb
melanocytes in vitiligo-associated human leukotrichia has not been formally
investigated, hair bulb melanocyte death occurs in C57BL/6Jler-vit/vit mice
[113]. Spontaneous (or induced) repigmentation is rare in human vitiligo; if it
occurs, the current consensus is that outer root sheath follicular melanocytes
have proliferated locally and entered the epidermis [102], as can be seen
following dermabrasion [36]. Others have suggested that melanocytes located in
the hair bulb can also repopulate vitiliginous epidermis [114]. However,
repigmentation may also occur via the epidermis itself because, despite the
current dogma, some poorly differentiated melanocytes are indeed retained in
lesional epidermis, even after vitiligo of long duration [115].
328 TOBIN
8.3
Circumscribed Poliosis
Poliosis is an inherited or acquired loss of pigment from a group of closely
positioned hair follicles resulting in a patch of white/hypopigmented hair.
Piebaldism, an autosomal dominant genetic disorder, is characterized by a white
forelock associated with diamond-shaped depigmentation on the forehead and is
associated with impaired melanocyte maturation/migration migration in affected
hair bulbs [116]. Focal mutations or deletions in the KIT gene encoding the
receptor for stem cell factor have been identified. Pigmentary defects in the
Waardenburg syndrome (types I–III) include partial or total heterochromia irides
and piebaldism, in addition to premature graying. Molecular studies of
Waardenburg (type I) syndrome also reveal mutations or deletions in the Kit
gene, whereas the MITF (microphthalmia transcription factor) gene is deleted in
Waardenburg type II syndrome [116]. Rare cases of poliosis have been described
with Tietz, Vogt-Koyanagi-Harada, Alezzandrini, Griscelli, and Apert
syndromes, as well as von Recklinghausen’s neurofibromatosis, and tuberous
sclerosis (Pringle syndrome). Reduced eumelanogenesis and melanocyte loss is
observed in Prader-Willi and Angelman syndromes, where deletions in the pink-
eyed dilution gene have been identified [116].
8.4
Genetic Poliosis
Albinism consists of a group of autosomal recessive diseases that exhibit a
congenital reduction or absence of melanin in skin, hair, and eyes [117]. The hair
and skin color can be affected to various degrees, ranging from complete white
hair to red, brown, and dark hair. The rare Chediak-Higashi syndrome is
additionally characterized by the production of silver gray/light blond hair on the
scalp.
While premature canities can appear without underlying pathology, it has also
been associated with pernicious anemia [118], hyper- or hypothyroidism,
osteopenia [97], and several rare syndromes (e.g.,Werner’s syndrome) [119].
CONCLUSION
The evolutionary selective pressures for hair pigmentation must have been
significant. Still, its value to modern humans, beyond social and sexual
communication, remains enigmatic. The distinct, but open, melanocyte
compartments in the epidermis and hair follicle attest to the bifunctionality of
melanocytes in the upper hair follicle; that is, these cells aid the repigmentation
of both epidermis and the new anagen hair bulb. In this way, this follicular
melanocyte population provides a very important reservoir function (e.g., after
epidermal injury). Moreover, amelanotic melanocytes in the outer root sheath, by
not devoting resources to melanin synthesis, remain plastic and are available to
participate in other, still poorly defined intercellular processes. Melanotic bulbar
melanocytes, on the other hand, appear to be largely devoted to producing very
large amounts of this melanin. However, the bioactive characteristics of melanin
beyond color are likely to be considerable, especially after transfer of melanin
granules to the hair-shaft-forming keratinocytes.
We are only beginning to unravel the mysteries of hair pigmentation in
humans. While geneticists continue to study pigmentation-associated gene
polymorphism that will help track human migrations throughout history, the
diversity of differentiation states exhibited by follicular melanocyte
subpopulations and the natural aging of these neural crest-derived cells continue
to offer the biologist new insights into the full potential of this part-time
pigmenter, immune cell, homeostasis regulator, and fulfiller of many other
functions.
ACKNOWLEDGMENTS
Some of the work described in this chapter was supported in part by grants from
Procter & Gamble Ltd., Surrey, England. This manuscript is dedicated to the
memory of Prof. Aodán S.Breathnach—mentor and friend.
REFERENCES
1. Morgan E. The Ascent of Woman, Souvenir Press, London 1985.
2. Bertazzo A, Costa C, Biasiolo M, Allegri G, Cirrincione G, Presti G. Determination
of copper and zinc levels in human hair: influence of sex, age, and hair
pigmentation. Biol Trace Elem Res 1996; 52:37–53.
3. Wood JM, Jimbow K, Boissy RE, Slominski A, Plonka PM, Slawinski J,
Wortsman J, Tosk J. What’s the use of generating melanin?. Exp Dermatol 1999; 8:
153–164.
4. Slominski A, Pawelek J. Animals under the sun: effects of ultraviolet radiation on
mammalian skin. Clin Dermatol 1998; 16:503–515.
5. Rees JL. The melanocortin 1 receptor (MCR1): more than just red hair. Pigment
Cell Res 2000; 13:135–140.
6. Rawles ME. Origin of pigment cells from the neural crest in the mouse embryo.
Physiol Zool 1947; 20:248–266.
7. Holbrook KA, Vogel AM, Underwood RA, Foster CA. Melanocytes in human
embryonic and fetal skin: a review and new findings. Pigment Cell Res (suppl)
1988; 1:6–17.
8. Jackson IJ. Molecular and developmental genetics of mouse coat color. Annu Rev
Genet 1994; 28:189–217.
9. Nakamura M, Tobin DJ, Richards-Smith B, Sundberg JP, Paus R. Mutant
laboratory mice with abnormalities in pigmentation: annotated tables. J Dermatol
Sci 2002; 28:1–33.
330 TOBIN
10. Fleischman RA, Gallardo T, Mi X. Mutations in the ligand-binding domain of the

kit receptor: an uncommon site in human piebaldism. J Invest Dermatol 1996; 107:
703–706.
11. Steel KP, Davidson DR, Jackson IJ. TRP2/DT, a new early melanocyte marker,
shows that steel growth factor (c-kit ligand) is a survival factor. Development
1992; 115:111–119.
12. Ito M, Kawa Y, Ono H, Okura M, Baba T, Kubota Y, Nishikawa SI, Mizoguchi M.
Removal of stem cell factor or addition of monoclonal anti-c-KIT antibody induces
apoptosis in murine melanocyte precursors. J Invest Dermatol 1999; 112:796–801.
13. Yoshida H, Kunisada T, Kusakabe M, Nishikawa S, Nishikawa SI. Distinct stages
of melanocyte differentiation revealed by analysis of non-uniform pigmentation
patterns. Development 1996; 122:1207–1214.
14. EM Peters, DJ Tobin, N Botchkareva, M Maurer, R Paus. C-kit expression in
developing hair follicles: Migration of melanoblasts into the developing murine
hair follicle is accompanied by transient c-kit expression. J Histochem Cytochem
2002; 50:751–766.
15. Iwamoto T, Takahashi M, Ohbayashi M, Nakashima I. The ret oncogene can
induce melanogenesis and melanocyte development inWv/Wv mice. Exp Cell Res
1992; 200:410–415..
16. Lahav R, Dupin E, Lecoin L, Glavieux C, Champeval D, Ziller C, Le Douarin NM.
Endothelin selectively promotes survival and proliferation of neural crest-derived
glial and melanocytic precursors in vitro. Proc Natl Acad Sci U S A 1998; 95:
14214–14219.
17. Hosoda K, Hammer RE, Richardson JA, Baynash AG, Cheung JC, Giaid A,
Yanagisawa M. Targeted and natural piebald-lethal mutations of endothelin-B
receptor gene produce megacolon associated with spotted coat color in mice. Cell
1994; 79:1267–1276.
18. Baynash AG, Hosoda K, Giaid A, Richardson JA, Emoto N, Hammer RE,
Yanagisawa M. Interaction of endothelin-3 with endothelin-B receptor is essential
for development of epidermal melanocytes and enteric neurons. Cell 1994; 79:
1277–1285.
19. Bedell MA, Largaespada DA, Jenkins NA, Copeland NG. Mouse models of human
disease. II: Recent progress and future directions . Genes Dev 1997; 11:11–43.
20. Shin MK, Levorse JM, Ingram RS, Tilghman SM. The temporal requirement for
endothelin receptor-B signaling during neural crest development. Nature 1999; 402:
496–501.
21. Perris R. The extracellular matrix in neural crest cell migration. Trends Neurosci
1997; 20:23–31.
22. Henderson DH, Copp AJ. Role of the extracellular matrix in neural crest cell
migration. J Anat 1997; 191:507–515.
23. Hirai Y, Nose A, Kobayashi S, Takeichi M. Expression and role of E- and P-
cadherin adhesion molecules in embryonic histiogenesis. II: Skin morphogenesis.
Development 1989; 105:271–277.
24. Chase HB, Rauch H, Smith VW. Critical stages of hair follicle development and
pigmentation in the mouse. Physiol Zool 1951; 25:1–19.
25. Hashimoto K. The ultrastructure of the skin of human embryos. VIII:Melanoblasts
and intrafollicular melanocytes. J Anat 1971; 108:99–108.
26. Tobin DJ, Paus R, Slominski A. The hair follicle pigmentary system. In: Paus R,
ed. The Biology of the Hair Follicle. Austin: RG Landes Co. In preparation.
27. Ito M. Unusual distribution of melanocytes in hair bulbs from a Negro subject.
Arch Dermatol Res 1991; 283:274–277.
28. Tobin DJ, Cargnello JA: Partial reversal of canities in a twenty-two year old
Chinese male. Arch Dermatol 1992; 129:789–791.
29. Tobin DJ, Fenton DA, Kendall MD. Ultrastructural observations on the hair bulb
melanocytes and melanosomes in acute alopecia areata. J Invest Dermatol 1990; 94:
803–807.
30. Vennegoor C, Hageman P, Van Nouhuijs H, Ruiter DJ, Calafat J, Ringens PJ, Rumke
P. A monoclonal antibody specific for cells of the melanocyte lineage. Am J Pathol
1988; 130:179–292.
31. Horikawa T, Norris DA, Johnson TW, Zekman T, Dunscomb N, Bennion SD,
Jackson RL, Morelli JG. DOPA-negative melanocytes in the outer root sheath of
human hair follicles express premelanosomal antigens but not a melanosomal
antigen or the melanosome-associated glycoproteins tyrosinase, TRP-1, and
TRP-2. J Invest Dermatol 1996; 106:28–35.
32. Tobin DJ, Bystryn JC. Different populations of melanocytes are present in hair
follicles and epidermis. Pigment Cell Res 1996; 9:304–310.
33. O’Sullivan JR, Williams A, Gummer C, Tobin DJ. Distribution of follicular
melanocytes during the hair growth cycle. J Invest Dermatol 2000; 114:861.
34. Grichnik JM, Ali WN, Burch JA, Byers JD, Garcia CA, Clark RE, Shea CR. KIT
expression reveals a population of precursor melanocytes in human skin. J Invest
Dermatol 1996; 106:967–971.
35. Tobin DJ, Colen SR, Bystryn JC. Isolation and long-term culture of human hair-
follicle melanocytes. J Invest Dermatol 1995; 104:86–89.
36. Staricco RG. Amelanotic melanocytes in the outer sheath of the human hair follicle
and their role in the repigmentation of regenerated epidermis. Ann NY Acad Sci
1963; 100:239–255.
37. Slominski A, Paus R, Plonka P, Chakraborty A, Maurer M, Pruski D, Lukiewicz S.
Melanogenesis during the anagen–catagen–telogen transformation of the murine
hair cycle. J Invest Dermatol 1994; 102:862–869.
38. Tobin DJ, Slominski A, Botchkarev V, Paus R. The fate of hair follicle
melanocytes during the hair growth cycle. J Invest Dermatol Symp Proc 1999; 4:
323–332.
39. Paus R. Immunology of the hair follicle. In: Bos JD . The Skin Immune System,
CRC Press, Boca Raton, FL 1997; 4:377–395.
40. Fitzpatrick TB, Breathnach AS. Das epidermale Melanin-Einheitsystem. Dermatol
Wochenschr 1963; 147:481–489..
41. Tobin DJ, Paus R. Graying: gerontobiology of the hair follicle pigmentary unit.
Exp Gerontol 2001; 36:29–54.
42. Slominski A, Paus R. Melanogenesis is coupled to murine anagen: towards new
concepts for the role of melanocytes and the regulation of melanogenesis in hair
growth. J Invest Dermatol 1993; 101:90S–97S.
43. Nordlund JJ, Ortonne J-P. The normal color of human skin. In: Nordlund JJ
Boissy, RE ,Hearing VJ, King RA, Ortonne J-P. The Pigmentary System:
Physiology and Pathophysiology, Oxford University Press, New York 1998; 101:
475–487.
332 TOBIN
44. Hara M, Yaar M, Byers HR, Goukassian D, Fine RE, Gonsalves J, Gilchrest BA.
Kinesin participates in melanosomal movement along melanocyte dendrites. J
45. Vancoillie G, Lambert J, Mulder A, Koerten HK, Mommaas AM, Van Oostveldt P,
Naeyaert JM. Kinesin and kinectin can associate with the melanosomal surface and
form a link with microtubules in normal human melanocytes. J Invest Dermatol
2000; 114:421–429.
46. Garcia RI. Ultrastructure of melanocyte-keratinocyte interactions. Pigment Cell
1979; 4:299–310.
47. Nascimento AA, Amaral RG, Bizario JC, Larson RE, Espreafico EM. Subcellular
localization of myosin-V in the B16 melanoma cells, a wild-type cell line for the
dilute gene. Mol Biol Cell 1997; 8:1971–1988.
48. Wei Q, Wu X, Hammer JA. The predominant defect in dilute melanocytes is in
melanosome distribution and not cell shape, supporting a role for myosin V in
melanosome transport. J Muscle Res Cell Motil 1997; 18:517–527.
49. Inazu M, MishimaY. Detection of eumelanogenic and pheomelanogenic
melanosomes in the same normal human melanocyte. J Invest Dermatol 1993; 100:
172S–175S.
50. Jimbow K, Park JS, Kato F, Hirosaki K, Toyofuku K, Hua C, Yamashita T.
Assembly, target-signaling and intracellular transport of tyrosinase gene family
proteins in the initial stage of melanosome biogenesis. Pigment Cell Res 2000; 13
(4): 222–229.
51. Kushimoto T, Basrur V, Valencia J, Matsunaga J, Vieira WD, Ferrans VJ, Muller
J, Appella E, Hearing VJ. A model for melanosome biogenesis based on the
purification and analysis of early melanosomes. Proc Natl Acad Sci U S A 2001; 98
(4): 10698–10703.
52. Schallreuter KU. A review of recent advances on the regulation of pigmentation in
the human epidermis. Cell Mol Biol (Noisy-le-Grand) 1999; 45(7): 943–949.
53. Napolitano A, Memoli S, Prota G. A new insight in the biosynthesis of
pheomelanins: characterization of a labile 1,4-benzothiazine intermediate. J Org
Chem 1999; 64:3009–3011.
54. Slominski A. Schallreuter KU. Control of follicular melanogenesis. In: Paus R, ed.
The Biology of the Hair Follicle. Austin: RG Landes Co. In preparation.
55. Snell RS. Hormonal control of hair color. In: V. Riley (ed) Pigmentation: Its
Genesis and Biologic Control., Meredith, New York 1972; 64:193–205.
56. Levine N, Sheftel SN, Eytan T, Dorr RT, Hadley ME, Weinrach JC, Ertl GA, Toth
K, McGee DL, Hruby VJ. Induction of skin tanning by subcutaneous
administration of a potent synthetic melanotropin. JAMA 1991; 266:2730– 2736.
57. Slominski A, Paus R, Constantino R. Differential expression and activity of
melanogenesis-related proteins during induced hair growth in mice.. J Invest
Dermatol 1991; 96:172–179.
58. Valverde P, Healy E, Jackson I, Rees JL, Thody AJ. Variants of the melanocyte-
stimulating hormone receptor gene are associated with red hair and fair skin in
humans. Nat Genet 1995; 11:328–330.
59. Schallreuter KU, Beazley WD, Hibberts NA, Tobin DJ, Paus R, Wood JM. Pterins
in human hair follicle cells and in the synchronized murine hair cycle. J Invest
Dermatol 1998; 111(4): 545–550.
60. Hockenbery DM, Oltvai ZN, Yin XM, Milliman CL, Korsmeyer SJ. Bcl-2 functions
in an antioxidant pathway to prevent apoptosis. Cell 1993 241–251.
61. Li G, Schaider H, Satyamoorthy K, Hanakawa Y, Hashimoto K, Herlyn M.
Downregulation of E-cadherin and desmoglein 1 by autocrine hepatocyte growth
factor during melanoma development. Oncogene 2001 8125–8135.
62. Reynolds J. The epidermal melanocytes of mice. J Anat 1954; 88:45–58.
63. Paus R, Stenn KS, Link RE. Telogen skin contains an inhibitor of hair growth. Br J
Dermatol 1990; 122:777–784.
64. Silver AF, Chase HB. DNA synthesis in the adult hair germ during dormancy
(telogen) and activation (early anagen). Dev Biol 1970; 21:440–451.
65. Slominski A, Paus R, Constantino R. Differential expression activity of
melanogenesis-related proteins during induced hair growth in mice. J Invest
Dermatol 1991; 96:172–179.
66. Sugiyama S, Saga K, Morimoto Y, Takahashi M. Proliferating activity of the hair
follicle melanocytes at the early anagen III stages in the hair growth cycle:
detection by immunocytochemistry for bromodeoxyuridine combined with DOPA
reaction cytochemistry. J Dermatol 1995; 22:396–402.
67. Jimbow K, Roth SI, Fitzpatrick TB, Szabo G. Mitotic activity in non-enoplastic
melanocytes in vivo as determined by histochemical, autoradiographic, and
electron microscopic studies. J Cell Biol 1975; 66:666–670.
68. Sugiyama S, Kukita A. Melanocyte reservoir in the hair follicles during the hair
growth cycle: an electron microscopicstudy. In: Biology and Disease of the Hair, K
Toda, Y Ishibashi, Y Hori, F Morikawa (eds.), University of Tokyo Press,Tokyo,
1976; 66:81–200.
69. Sugiyama S. Mode of re-differentiation and melanogenesis of melanocytes in
murine hair follicle. J Ultrastruct Res 1979; 67:40–54.
70. Weedon D, Strutton G. Apoptosis as the mechanism of the involution of hair
follicles in catagen transformation. Acta Dermatol Venereol (Stockh) 1981; 61:335–
359.
71. Lindner G, Botchkarev VA, Botchkareva NV, Ling G, van der Veen C Paus R.
Analysis of apoptosis during hair follicle regression (catagen). Am J Pathol 1997;
151:1601–1617.
72. Grichnik JM, Ali WN, Burch JA, Byers JD, Garcia CA, Clark RE, Shea CR. KIT
expression reveals a population of precursor melanocytes in human skin. J Invest
Dermatol 1996; 106:967–971.
73. Botchkareva NV, Khlgatian M, Longley BJ, Botchkarev VA, Gilchrest BA. SCF/c-
kit signaling is required for cyclic regeneration of the hair pigmentation unit.
FASEB J 2001; 15:645–658.
74. Commo S, Bernard BA. Melanocyte subpopulation turnover during the human hair
cycle: an immunohistochemical study. Pigment Cell Res 2000; 13: 253–259.
75. Tobin DJ, Hagen E, Botchkarev VA, Paus R. Do hair bulb melanocytes undergo
apoptosis during hair follicle regression (catagen)? J Invest Dermatol 1998; 111:6;
941–947.
76. Christoph T, Muller-Rover S, Audring H, Tobin DJ, Hermes B, Cotsarelis G,
Ruckert R, Paus R. The human hair follicle immune system: cellular composition
and immune privilege. Br J Dermatol 2000; 142:862–873.
77. Tobin DJ. A possible role for Langerhans cells in the removal of melanin from
early catagen hair follicle. Br J Dermatol 1998; 138:795–798.
334 TOBIN
78. Niedecken HW, Lutz G, Bauer R, Kreysel HW. Expression of Langerhans cell
antigens in the hair follicles in alopecia areata. In: Van Neste D , Lachapelle JM,
Antoine JL. Trends in Human Hair Growth and Alopecia Research, Kluwer
Academic Publishers, Dordrecht 1989; 138:291–298.
79. Whiteman DC, Parsons PG, Green AC. Determinants of melanocyte density in
adult human skin. Arch Dermatol Res 1999; 291:511–516.
80. Medrano EE, Yang F, Boissy R, Farooqui J, Shah V, Matsumoto K, Nordlund JJ,
Park HY. Terminal differentiation and senescence in the human melanocyte:
repression of tyrosine-phosphorylation of the extracellular signal-regulated kinase 2
selectively defines the two phenotypes. Mol Biol Cell 1994; 5: 497–509.
81. Bennett DC. Differentiation in mouse melanoma cells: initial reversibility and an
on-off stochastic model. Cell 1983; 34:445–453.
82. Ames BN, Shigenaga MK, Hagen TM. Oxidants, antioxidants, and the
degenerative process of aging. Proc Natl Acad Sci U S A 1993; 90:7915–7922.
83. Clampisi J. The role of cellular senescence in skin aging. J Invest Dermatol Symp
Proc 1998; 3: l-5.
84. Hegedus ZL. The probable involvement of soluble and deposited melanins, their
intermediates and the reactive oxygen side-products in human diseases and aging.
Toxicology 2000; 14:145/2–3): 85–101.
85. Keogh EV, Walsh RJ. Rate of graying of human hair. Nature 1965; 207: 877–878.
86. Ibrahim L, Wright EA. The long term effect of repeated pluckings on the function
of the mouse vibrissal follicles. Br J Dermatol 1978; 99:371–376.
87. Orfanos CE. Das weisse Haardes alterer Menschen. Arch Klin Exp Dermatol 1970;
236:368–384.
88. Toyoda M, Morohashi M. Morphological alterations of epidermal melanocytes in
photoaging: an ultrastructural cytomorphometric study. Br J Dermatol 31998; 139:
444–452.
89. Sato S, Kukita A, Jimbow K. Electron microscopic studies of dendritic cells in the
human gray and white matrix during anagen. Pigment Cell 1973; 1:20–26.
90. Weisse I. Changes in the aging rat retina. Ophthalm Res 1995; 27: Suppl 1: 154–
163.
91. Bowers R, Chun DQ. Ultrastructural study of senescence of regenerating feather
melanocytes in the jungle fowl. In: Bagnara K, Schartl M. Biological, Molecular
and Clinical Aspects of Pigmentation—Pigment Cell, University of Tokyo
Press,Tokyo 1985; 1:347–357.
92. Westerhof W, Njoo D, Menke KE. Miscellaneous hypomelanoses: disorders
characterized by extracutaneous loss of pigmentation. In: Nordlund JJ, Boissy RE,
Hearing VJ, King RA, Ortonne J-P. The Pigmentary System: Physiology and
Pathophysiology, Oxford University Press, New York 1998; 1: 475–487.
93. Nag1 W. Different growth rates of pigmented and white hair in the beard:
differentiation vs.proliferation?. Br J Dermatol 1995; 132:94–97.
94. Slominski A, Paus R, Schadendorf D. Melanocytes as “sensory” and regulatory
cells in the epidermis. J Theor Biol 1993; 164:103–120.
95. Tang A, Eller MS, Hara M, Yaar M, Hirohashi S, Gilchrest BA. E-cadherin is the
major mediator of human melanocyte adhesion to keratinocytes in vitro J Cell Sci
1994; 107:983–992.
96. D’Agostini F, Balansky R, Pesce C, Fiallo P, Lubet RA, Kelloff GJ, De Flora S.
Induction of alopecia in mice exposed to cigarette smoke. Toxicol Lett 2000; 114:
1–3,117–123.
97. Rosen CJ, Holick MF, Millard PS. Premature graying of hair is a risk marker for
osteopenia. J Clin Endocrinol Metab 1994; 79:854–857.
98. Eisenstein I, Edelstein J. Gray hair in black males a possible risk factor in coronary
artery disease. Angiology 1982; 33(10): 652–654.
99. Correa A, Mohan A, Jackson L, Perry H, Helzlsouer K. Use of hair dyes,
hematopoietic neoplasms, and lymphomas: a literature review. I: Leukemias and
myelodysplastic syndromes. Cancer Invest 2000; 8(4): 366–380.
100. Correa A, Jackson L, Mohan A, Perry H, Helzlsouer K. Use of hair dyes,
hematopoietic neoplasms, and lymphomas: a literature review. II: Lymphomas and
multiple myeloma. Cancer Invest 2000; 18(5): 467–479.
101. Xie Z, Hayakawa R, Sugiura M, Kojima H, Konishi H, Ichihara G,Takeuchi Y.
Experimental study on skin sensitization potencies and cross-reactivities of hair-
dye-related chemicals inguineapigs. Contact Dermatitis 2000; 42(5): 270–275.
102. Cui J, Shen LY, Wang GC. Role of hair follicles in the repigmentation of vitiligo. J
103. Horikawa T, Norris DA, Yohn JJ, Zekman T, Travers JB, Morelli JG. Melanocyte
mitogens induce both melanocyte chemokinesis and chemotaxis. J Invest Dermatol
1995; 104(2): 256–259.
104. Halaban R, Langdon R, Birchall N, Cuono C, Baird A, Scott G, Moellmann G,
McGuire J. Basic fibroblast growth factor from human keratinocytes is a natural
mitogen for melanocytes. J Cell Biol 1988; 107(4): 1611–1619.
105. Imokawa G, Yada Y, Miyagishi M. Endothelins secreted from human keratinocytes
are intrinsic mitogens for human melanocytes. J Biol Chem 1992; 267(34): 24675–
24680.
106. Jordan SA, Jackson IJ. MGF (KIT ligand) is a chemokinetic factor for melanoblast
migration into hair follicles. Dev Biol 2000; 225(2): 424–436.
107. Shetty M. Radiation therapy activates melanocytes in hair. Br Med J 1995; 311:
1582.
108. Verbov J. Erosive candidiasis of the scalp, followed by the reappearance of black
hair after 40 years. Br J Dermatol 1981; 105:595–598.
109. Goldenhersh MA. Rapid whitening of the hair first reported in the Talmud.
Possible mechanisms of this intriguing phenomenon. Am J Dermatopathol 1992;
14:367–368.
110. Tobin DJ. Morphological analysis of hair follicles in alopecia areata. Microsc Res
Techniques 1997; 38:443–451.
111. Bystryn J-C, Tobin DJ. Alopecia areata is associated with antibodies to hair follicle
melanocytes. J Invest Dermatol 1994; 102:4; 532.
112. Paus R, Slominski A, Czarnetzki BM. Alopecia areata: an autoimmune-response
against melanogenesis-related proteins, exposed by abnormal MHC class I-
expression in the proximal anagen hair bulb? Yale J Biol Med 1993; 66: 541–554.
113. Boissy RE, Moellmann GE, Lerner AB. Morphology of melanocytes in hair bulbs
and eyes of vitiligo mice. Am J Pathol 1987; 127:380–238.
114. Arrunategui A, Arroyo C, Garcia L, Covelli C, Escobar C, Carrascal E, Falabella R.
Melanocyte reservoir in vitiligo. Int J Dermatol 1994; 33:484–487.
336 TOBIN
115. Tobin DJ, Swanson NN, Pittelkow MR, Peters EMJ, Schallreuter KU. Melanocytes
are not absent in lesional skin of long duration vitiligo. J Pathol 2000; 191:1–10.
116. Spritz RA. Piebaldism, Waardenburg syndrome, and related disorders of
melanocyte development. Semin Cutan Med Surg 1997; 16:15–23.
117. Oetting WS. Albinism. Curr Opin Pediatr 1999; 11:565–571.
118. Noppakun N, Swasdikul D. Reversible hyperpigmentation of skin and nails with
white hair due to vitamin B12 deficiency. Arch Dermatol 1986; 122(8): 896–899.
119. Abe T, Yamaguchi Y, Izumino K, Ozaki M, Yamakawa K, Kondo H, Sera Y,
Uotani S,Takino H, Kawasaki E, Yamasaki H, Eguchi K. Evaluation of insulin
response in glucose tolerance test in a patient with Werner’s syndrome: a 16-year
follow-up study. Diabetes Nutr Metab 2000; 13:113–118.
14
Androgen Influence on Hair Growth
Valerie Anne Randall
University of Bradford, Bradford, United Kingdom
The hair follicle, a highly unusual, dynamic organ found only in mammalian
skin, has contributed greatly to the evolutionary success of mammals. Each follicle
synthesizes and supports a hair and, most excitingly, possesses the ability to
partially recapitulate embryogenesis to replace this hair during the hair growth
cycle [1, 2]. The new hair can resemble the previous one or differ from it in size
and/or color. This is seen very dramatically in Scottish mountain hares, whose
thick white winter coat markedly contrasts with the shorter brown summer
version [3]. Major changes also occur in human hair growth, mainly under the
influence of androgens; around and after puberty, follicles that produced tiny,
almost transparent vellus hairs in childhood form larger, thicker and darker
terminal hairs in many areas such as the axilla in both sexes and the face of boys
[4–6]. To achieve this, hair follicles pass through regular three-phase growth
cycles: (1) development and growth (anagen), when the lower follicle is
regenerated, forming a new hair that generally replaces the original hair; (2)
regression (catagen), when growth stops, the lower follicle is resorbed, and the
whole hair becomes keratinized; and (3) rest (telogen), when the hair normally
remains in the skin until replaced by the next hair. An additional process of
exogen, involving the active release of the old hair, has also recently been
proposed [7].
This highly unusual facility of regularly generating new hairs has contributed
to the ability of mammals to adapt to different climatic conditions. Insulation and
camouflage, two important functions of mammalian hair, may have constant
requirements in the tropics, but in the temperate and arctic zones adaptation to
environmental changes is essential for survival. The hair growth cycle also
enables replacement of damaged hairs; this is important for protective hairs, or
those acting as neuroreceptors, in all species (including human eyelashes,
eyebrows, and scalp hair). In addition, the ability to change the type of hair
facilitates the role of hairs in social and sexual communication, enabling
different hair colors or markings to distinguish between young and mature
mammals (e.g., pubic and axillary hair) and between the sexes of adults (e.g.,
manes of male lions and red deer and the human beard). Hormones coordinate
these hair changes with alterations in the external environment or the
individual’s age and sex [8]. However, the response of individual follicles is
338 RANDALL
specific to their body site; beard follicle response to the same androgens clearly
differs from those on the same individual’s head. This chapter reviews our
current understanding of the hormonal regulation of hair growth, particularly the
influence of androgens on human hair follicles, and the mechanisms of how it
occurs.
1
EXPERIMENTAL APPROACHES
There have been extensive investigations into the hormonal control of hair
growth, and this is still a major focus for research. Initial studies focused on
classical observations of hair growth under normal or manipulated conditions in
people [1, 10] and other mammals [8]. Important information about the
mechanism of action of androgens in human hair follicles has also been gained
by investigating genetic abnormalities of hair growth (discussed later).
Current approaches focus on investigating the identity of paracrine factors in
follicles by using primary cultured follicular cells, particularly those of the
dermal papilla [11, 12], cultured isolated whole follicles, and genetically [13, 14]
manipulated animals, relying particularly on conditional gene targeting of mice
(limited expression knockout models) [15]. At the moment cultured dermal
papilla cells derived from follicles with differing responses to androgens appear
to be the most useful route for investigating androgen action. A recent approach
linking human media from balding dermal papilla cells to a delay in hair growth
in mice opens exciting new possibilities [16].
2
SEASONAL CHANGES IN HAIR GROWTH
Changes in hair growth during the year in many mammals are closely
coordinated with the length of daylight, and to a smaller extent with temperature,
in a similar manner to the seasonal breeding activity that occurs in many temperate
mammals (reviewed in Ref. [8]). Environmental cues are interpreted for the hair
follicle by alterations in the pineal and hypothalamuspituitary hormones, which
regulate the levels of many circulating hormones, including gonadol, thyroid, and
corticosteroid hormones, known to alter mammalian follicular activity [8]. The
pituitary hormone prolactin has also been strongly implicated in seasonal
changes in red deer [17, 18], mink [19], and goats [20].
Although less obvious than in many other mammals, growth of human hair
also undergoes quite marked seasonal variation [21–23]. Scalp hair in normal
Caucasian men and women shows a single annual cycle with a pronounced
shedding in the autumn [21, 22]. This was confirmed by assessing the percentage
of hairs in anagen throughout the year. This reached a maximum of over 90% in
the early spring, falling to about 80% in late summer [22]. Since most
nonbalding scalp follicles are in anagen for more than 2 to 3 years, such a
ANDROGEN INFLUENCE ON HAIR 339
marked seasonal effect suggests strong controlling influences coordinating those

follicles in the later parts of anagen. The subjects, all men with indoor
occupations, showed pronounced increases in the amount of time they spent
outdoors in the longer days of summer: that is, they were exhibiting seasonal
changes of behavior related to the photoperiod. The hormones involved are not
yet known but this scalp follicle response may well be a reaction to hormones
similar to those seen in other mammals.
A different pattern of seasonal effects was seen in the same men in the beard
and thigh regions, where the hair is a secondary sexual characteristic and men
have much larger hairs than women [22]. Beard and thigh hair growth was low in
winter but increased in the summer in the case of the beard by more than 50%
(Fig. 1). This change may be facilitated by the increased summer androgen levels
reported in small studies of European men [24– 26], since these hair follicles are
considered to be androgen target hormones (discussed later).
3
FUNCTIONS AND PATTERNS OF HUMAN HAIR
GROWTH
At first sight human hair growth does not show too close a resemblance to that of
most other mammals, despite the widespread distribution of hair follicles on our
body surface and the seasonal variation in our hair growth. Human hair has lost
many functions seen in other mammals, including insulation and camouflage,
and therefore does not need to cover the skin. It does retain protective roles in
some sites. These include the eyelashes and eyebrows and probably scalp hair,
which may prevent sunburn and physical damage to the scalp and back of the
neck [27, 28], as well as heat loss in the winter, since there is little adipose tissue
in the scalp [27]. Follicles in these regions produce significant terminal hairs in
childhood, emphasizing their protective role.
The majority of other follicles form only vellus hairs in childhood, but during
puberty terminal hairs develop slowly in the axilla and pubic regions of both
sexes [5–7]. This clearly signals the move from childhood into the adult world.
In addition, adult maleness is clearly distinguished by beard growth on the face
and terminal hair on the chest, upper pubic triangle and upper arms and legs [6].
(Fig. 2 upper panel).
These male-specific changes develop initially during puberty, but the changes
are gradual and hair growth continues to increase in many regions for many
years. Beard growth is produced quite rapidly during puberty but continues to
rise until the mid-30s [29], while terminal hair on the chest or ear canal may not
appear until some years after puberty [30].
This role of hair in human social and sexual communication is very important
whatever the genetic or cultural background, although its signifi-cance is not
always appreciated. A head covered with hair in good condition is generally
recognized as a symbol of health and, therefore, forms part of an individual’s
340 RANDALL
FIGURE 1 Androgen-dependent hair growth shows significant seasonal changes during

the year. The rate of beard (top diagram) and thigh (bottom diagram) hair growth is faster
in the summer in Caucasian men aged 18 to 39 living in northern England. (Data redrawn
from Ref. 22, reproduced from VA Randall. Androgens: The main regulator of human
hair growth. In: FM Camacho, VA Randall, VH Price, eds. Hair and Its Disorders:
Biology, Pathology and Management. London: Martin Dunitz, 2000, pp 69–82.)
attractiveness. Hair is also used to express particular views in many societies.

This ranges from the shaved heads of Christian and Buddhist monks to the
traditional short haircuts of western soldiers to the uncut hair of Sikhs. Similarly,
since the beard can be used in male threatening display behavior, the custom of
shaving off the beard each day by most western men may enable greater
cooperation among men living and working so closely together [27].
This importance of hair in communication explains the psychological distress
undergone by sufferers of hair disorders such as hirsutism, male pattern hair
growth in women (Fig. 2, lower panel), or alopecia (hair loss). A gradual,
progressive loss of hair on the scalp is a common feature in many men, causing
male pattern baldness or androgenetic alopecia. This occurs in a relatively
precise pattern, with gradual regression of the frontal hairline and thinning on the
vertex [9, 31], which in extreme cases may leave terminal hair only around the
sides and back of the head (Fig. 3, upper panels). Although some women may
exhibit this type of hair loss, it is unusual. Women more often have a diffuse loss
of hair on the crown with retention of the frontal hair line [32, 33] (Fig. 3, lower
panel). Regardless of whether this common pronounced hair loss in men
originally had a biological role such as marking the older male leader (reviewed
in Ref. 34), hair loss now can cause psychological distress and a reduction in the
quality of life (reviewed in Ref. 34), even among those who have never consulted
anyone for treatment [35]. Since neither hirsutism nor male pattern baldness is
currently easily controlled, there is a need for further understanding of these
disorders.
4
THE RESPONSE OF HUMAN HAIR FOLLICLES TO
ANDROGENS DEPENDS ON THEIR BODY SITE
During and after puberty, androgens are the main regulator of the changes in
human hair growth just discussed. During puberty the changes in both sexes
parallel the rises in plasma androgens, occurring later in boys than girls [36, 37],
and castration inhibits both beard formation [29] and male pattern baldness [38, 39].
The strongest support for the role of androgens comes from individuals with
complete androgen insufficiency (i.e., without functional androgen receptors)
[39]. These XY individuals develop a feminine-type phenotype, even if their
testes are not removed and their circulating androgens are high, but they do not
produce the adult pattern of terminal hair of either sex and do not develop male
pattern baldness (Fig. 2, lower panel).
At the same time, human hair growth illustrates a biological paradox of
hormone action (Fig. 4). Androgens are stimulating vellus follicles to change to
terminal ones in many areas after puberty, while at the same time, often in the
same individual, they are also stimulating the reverse transformation of large
terminal follicles to tiny vellus ones on the scalp. Interestingly, they appear to be
having no effect on other follicles such as the eyelashes (see Fig. 3).
This means that the response to androgens is intrinsic to the individual follicle.
In addition to these major differences in type of response, there is variation in
sensitivity among follicles that have the same stimulation or inhibition reaction.
For example, regression in androgenetic alopecia occurs in a progressive pattern
[9, 31] (Fig. 3), and in both young men and hirsute women, pigmented facial hair
develops initially above the mouth and center of the chin before expanding
across the face [4, 6]. In addition, although female levels of androgens promote
axillary and the female pubic pattern of terminal hair, the characteristic male
patterns normally require adult male levels [5, 6, 36, 37].
342 RANDALL
FIGURE 2 Human hair growth varies with different endocrine conditions. The small
amount of protective terminal hairs on the scalp, eyelashes, and eyebrows seen in children
is increased during puberty by axilliary and pubic hair growth in both sexes, plus beard,
chest and greater body hair in men (top diagram). None of this occurs without functional
androgen receptors, and only axillary and pubic hair is formed in the absence of 5 -
reductase type 2 (bottom diagram). In genetically predisposed individuals, androgens may
also cause inhibition of scalp hair growth, particularly in men. Raised circulating
androgens or idiopathic causes (e.g., increased follicle sensitivity) can also lead to
hirsutism (i.e., male pattern hair distribution in women) (bottom diagram). (Reproduced
from VA Randall. Androgens: The main regulator of human hair growth. In: FM
Camacho, VA Randall, VH Price, eds. Hair and Its Disorders: Biology, Pathology and
Management. London: Martin Dunitz, 2000, pp 69–82.)
FIGURE 3 The pattern of hair loss in androgenetic alopecia. Androgens cause a gradual
inhibition of hair growth on the scalp in genetically predisposed individuals; this is much
more common in men than women. In men, the first signs are generally temporal
regression, which spreads backward and joins thinning regions on the vertex to give a bald
crown (top diagram). (After Ref. 9). In women, the pattern of androgen-dependent hair
loss (bottom diagram) differs from that in men with the front hairline normally being
retained and a general thinning on the vertex gradually becoming more pronounced until
the vertex becomes bald. (After Ref. 32.)
Clearly each follicle has a genetically programmed response to androgens.

This is presumably due to changes in the genes that can be expressed in any
particular follicle during their development. This view is supported by the
observation that in the chick the dermis of the frontal-parietal scalp, which
corresponds to the balding regions in human scalp, develops from the neural crest,
while the occipital-temporal region, which is “nonbalding” in men, arises from
the mesoderm [40]. The concept of hair transplant surgery to correct
344 RANDALL
androgenetic alopecia is based on this end-organ specificity of individual

follicles [41]. Hair follicles from the “nonbalding” regions of the scalp such as
the nape of the neck are transplanted to balding regions. Once established, they
retain their genetic lack of androgen response and remain as terminal follicles
while minaturization of any remaining natural follicles from the region continues.
Although androgens are required to transform follicles to produce a different
type of hair, their role in maintaining the effect is less clear. In men who are
castrated after puberty, neither beard growth [29] nor androgenetic alopecia [42]
returns to prepubertal levels, suggesting that at least some of the altered gene
expression does not need androgen to be maintained. However, male beard
growth is enhanced in the summer, presumably by fluctuations in hormone levels
[22] (Fig. 1), the antiandrogen cyproterone acetate stimulates regression of
hirsutism [43], and the 5 -reductase inhibitor finasteride may cause regrowth of
androgenetic alopecia [44]. These results indicate a requirement for androgens to
maintain the status quo as well as to stimulate progression of androgenic
responses. An explanation for the differences between these observations could
be the length of time that a follicle had been responding to androgens. For
example, if in long-standing alopecia fibrosis had occurred below the follicle
[45], then the follicle would seem unlikely to be able to re-form a terminal hair.
The difference in responses to androgens by follicles from various body sites
has an important consequence for those wishing to study the mechanism of
androgen action in human hair follicles. It is essential to carry out investigations
into follicles that do respond to androgen in vivo in the way that is relevant to the
project. Unfortunately, this means that the normally most available tissue,
nonbalding scalp hair follicles, is not an appropriate material to use for many
experiments.
5
HOW DO ANDROGENS ACT ON HAIR FOLLICLES?
5.1
The Mechanism of Androgen Action in Hair Follicles
Like other steroid hormones, androgens act on target cells by diffusing through
the plasma membrane and binding to specific intracellular receptors. On binding
the relevant steroid, the receptor complexes undergo a conformational change,
exposing DNA binding sites and, often in association with accessory proteins,
binding to specific hormone response elements (HREs) in the DNA, which
promotes the expression of specific hormone regulated genes (Fig. 5) [39]. The
mechanism for androgens is more complex than the mechanisms of other
steroids. In some tissues, such as skeletal muscle, testosterone, the main
circulating androgen in men, does bind to the receptor. However, in many tissues,
including the secondary sexual tissues such as the prostate, testosterone is
FIGURE 4 The differing effects of androgens on human hair follicles. After puberty,
androgens stimulate the gradual production of terminal hair in many regions (e.g., beard,
axilla, pubis) that during childhood formed only small, fine vellus hairs (top). Some
follicles producing terminal hair in childhood remain unaltered (e.g., eyelashes,
nonbalding scalp) (center). In genetically predisposed people, androgens may cause the
opposite gradual transformation of terminal to vellus follicles at the same time, causing
balding (bottom). (Reproduced from VA Randall. Androgens: the main regulator of
human hair growth. In: FM Camacho, VA Randall, VH Price, eds. Hair and Its Disorders:
Biology, Pathology and Management. London: Martin Dunitz, 2000, pp 69–82.)
346 RANDALL
metabolized intracellularly by the enzyme 5 -reductase to 5 -

dihydrotestosterone, a more potent androgen, which binds preferentially and
more strongly to the androgen receptor to activate gene expression (reviewed in
Ref. 46) (Table 1, Fig. 5).
All androgen-dependent hair follicles require the androgen receptor to respond
as demonstrated by the absence of adult hair characteristics in complete androgen
sensitivity (Fig. 2, lower panel), the natural human “knock-out” model for the
androgen receptor [39]. In contrast, the requirement for 5 -reductase appears to
vary with the follicle site. Individuals with 5 -reductase type 2 deficiency
produce only female patterns of pubic and axillary hair growth after puberty,
although their body shapes become masculinized [47] (Fig. 2, lower panel). This
suggests that 5 -dihydrotesto-sterone is necessary for all the male-speciflc hair
follicles including the beard, chest, and upper pubic triangle, while testosterone
itself can stimulate the follicles of the axilla and lower pubic triangle
characteristic of adult women. People with 5 -reductase deficiency have not
been reported to show androgenetic alopecia. Combined with the success of
finasteride, a 5 -reductase type 2 inhibitor, in restoring hair growth in some
cases of male pattern baldness [44], this suggests that 5 -reductase type 2 is also
important for androgen-related balding.
TABLE 1 Targets for Androgens in the Hair Follicle

Cell type Function Androgen actiona
Dermal papilla cells Regulation Direct (+ possibly indirect)
Follicular keratinocytes Form hair and outer and Indirect
inner root sheaths
Connective tissue sheath Synthesize connective Direct
cells tissue sheath
Melanocytes Synthesize and transfer Indirect
pigment
Endothelial cells Blood supply Indirect
alndirect actions are translated by paracrine factors after direct androgen effects on dermal
papilla cells (see Fig. 6).

FIGURE 5 The mechanism of androgen action. Androgens, like other steroids, diffuse from the blood through the plasma membrane. Inside
the cell, testosterone itself may bind to the specific, intracellular androgen receptor, or it may be metabolized to the more potent androgen 5 -
dihydrotestosterone by the enzyme 5 -reductase. The receptor binds 5 -dihydrotestosterone more strongly than testosterone. The hormone-
androgen receptor complexes undergo a change in shape, exposing their DNA binding sites. This enables binding to specific hormone response
elements (HREs) in the DNA, altering the expression of particular androgen-dependent genes and hence the production of specific proteins.
Androgen receptors are required for all human body hair growth and male pattern baldness, but 5 -reductase type 2 is not required for pubic
ANDROGEN INFLUENCE ON HAIR
and axillary hair growth. (Reproduced from VA Randall. Androgens: The main regulator of human hair growth. In: FM Camacho, VA Randall,
VH Price, eds. Hair and Its Disorders: Biology, Pathology, and Management. London: Martin Dunitz, 2000, pp 69–82.)
347
348 RANDALL
FIGURE 6 The current hypothesis for androgen action in the hair follicle. In this model, androgens enter the hair follicle via capillaries in the
follicle dermal papilla. After binding to androgen receptors in the dermal papilla cells of androgen-sensitive follicles, they stimulate an
alteration in the production of regulatory paracrine factors. These paracrine factors then alter the activity of other follicular cells including
keratinocytes and melanocytes. T, testosterone,?, unknown paracrine factors. (Reproduced from VA Randall. Androgens and hair. In: E
Nieschlag, HM Behre, eds. Testosterone: Action, Deficiency, Substitution, 2nd ed. Heidelberg, Germany: Springer-Verlag, 1998, pp 167–186.)
5.2
Current Model for the Mode of Action of Androgens in
Hair Follicles
The current hypothesis for the way in which androgens act on the hair follicle
focuses on the dermal papilla. The mesenchyme-derived dermal papilla, situated
at the base of the mainly epithelial hair follicle, plays an important regulatory
role, altering many parameters of the hair follicle and determining the type of
hair produced [48]. Since hair follicles appear to partially recapitulate
embryogenesis during the hair cycle and steroids act via the mesenchyme in
many developing steroid-dependent tissues (reviewed in Ref. 49), androgens
were proposed to act on epithelial and melanocyte components of the follicle
indirectly via the cells of the dermal papilla [49].
In this hypothesis, summarized in Figure 6, androgens enter the dermal papilla
via the blood capillaries, where they bind to androgen receptors in the dermal
papilla cells of androgen-dependent follicles. As discussed earlier, whether they
would be metabolized to 5 -dihydrotestosterone would depend on the site of the
follicle. For example, beard cells would first metabolize testosterone with 5 -
reductase type 2, but axillary and pubic cells would not. The next stage would be
the alteration of the dermal papilla cell’s gene expression by the androgen-
receptor complex to change their production of paracrine regulatory factors for
the other follicular cell types. These factors could be soluble growth factors and/
or extracellular matrix proteins [49, 50]. Extracellular matrix components are
also likely to be involved because the size of the hair produced by the follicle is
related to dermal papilla size, most of which is contributed by extracellular
matrix components [51].
Potential androgen targets in the follicle include the follicular keratinocytes
(epithelial cells), which divide and mature to form the hair and its inner and
outer root sheaths; the melanocytes, which alter the amount of pigment produced
under different endocrine conditions; the follicular endothelial cells (since larger
hair follicles will require a greater blood supply); and the follicular connective
tissue, or dermal, sheath, which surrounds the follicle and must accommodate
changes in size. The cells of the dermal papilla itself are also a target, since dermal
papilla size must alter to change the follicle and hair size [51]. A system in which
most of these were coordinated through the dermal papilla seems to represent a
realistic way to ensure the smooth coordination of the complex cell biological
responses needed to change a hair follicle’s size and function. In this hypothesis
the message from the circulating androgens would be interpreted by the dermal
papilla cells and transmitted to other target cells via paracrine factors.
350 RANDALL
FIGURE 7 Cultured dermal papilla cells from androgenetic alopecia hair follicles contain
higher levels of androgen receptors than nonbalding scalp cells (A), but the receptors
appear to be similar (B). Androgen receptor content was assessed in dermal papilla cells
from nonbalding and balding follicles by 2hr incubation with a range of concentrations of
[3H] mibolerone with or without 200×excess unlabeled steroid, in medium 199
supplemented, with 10% dextran-coated, charcoal-stripped serum. Cells had been
incubated for 24 hr in similar medium to reduce endogenous androgens. The specificity of
the receptor binding was compared by determining the effectiveness of various
androgens, the antiandrogen cyproterone acetate, and other types of steroid hormone at
the same concentration (100nM) to compete with 1 nM [3H] mibolerone under similar
conditions. The specificity of binding to receptors from balding cells was very similar to
that from nonbalding cells. EtOH, ethanol vehicle alone; DHT, 5 -dihydrotestosterone;
Mib, mibolerone; test, testosterone; Cyp, Cyproterone acetate; oest, estradiol; Prog,
progesterone; TMA, triamcinolone acetonide; HC, hydrocortisol. (Data from Ref. 53,
diagram reproduced from VA Randall. The biology of androgen alopecia. In: FM
Camacho, VA Randall, VH Price, eds. Hair and Its Disorders. Biology, Pathology, and
Management. London: Martin Dunitz, 2000, pp 123–126.)
5.3
Support for the Dermal Papilla Model
The dermal papilla model has received considerable experimental support.
Specific high-affinity, low-capacity androgen receptors have been identified in
cultured dermal papilla cells derived from androgen target follicles such as
human beard [52] and balding scalp [53] (Fig. 7) and red deer mane [54].
Androgen receptors have also been localized by immunohistochemistry in the
dermal papilla cells of hair follicles, although the distribution in the epithelial
components varies with the type of androgen receptor antibody used. Monoclonal
antibody studies have revealed no epithelial cell staining in human [55, 56] or
red deer mane [54] follicles, but a study using a polyclonal antibody reported
staining in the outer root sheath [57]. This latter result is difficult to interpret;
however, it may be a methodological artifact—in my experience, polyclonal
antibodies for many factors stain differentiated follicular keratinocytes. Further
important support comes from studies of testosterone metabolism by cultured
dermal papilla cells. Beard cells metabolize testosterone to 5 -
dihydrotestosterone [58, 59], which is retained intracellularly [59], indicating
binding to the androgen receptor. In contrast, neither pubic nor axillary cells
contained significant amounts of 5 -dihydrotestosterone intracellularly, even
when cultured for 24 hr (Fig. 8) [60], suggesting that androgen action does not
require metabolism to 5 -dihydrotestosterone. The most important aspect of
these in vitro results is their parallel with the absence of beard growth, but
presence of axillary growth and a female pubic pattern, in patients with 5 -
reductase deficiency [47] (Fig. 2, lower panel).
The third component of this model is the altered production of paracrine
factors by dermal papilla cells under androgen stimulation. Several bioassays
have been used to investigate the production of mitogenic factors by either
coculturing dermal papilla cells with other cells or collecting “conditioned
media” (i.e., media in which dermal papilla cells have been grown) and assessing
the ability of this material to promote growth in other cells. These bioassays have
confirmed that human dermal papilla cells secrete soluble, proteinaceous factors
able to stimulate growth in other dermal papilla cells [50, 61], outer root sheath
cells [56, 62], and transformed epidermal keratinocytes [63]. These results, plus
their ability to produce vascular endothelial factor (VEGF) [64], an important
regulator of endothelial cells, and stem cell factor (SCF, ckit ligand, Steel factor,
mast cell growth factor) [64], an important factor for melanocytes, suggests that
dermal papilla cells can regulate the activity of all these cell types.
Interestingly for future experiments, at least some of these mitogenic factors
can cross the species divide between human beings and rodents as human dermal
papilla cell media can stimulate the growth of rat whisker cells [16]. When the
effect of androgens was assessed, testosterone was found to have increased the
mitogenic capacity of beard cells to stimulate outer root sheath cells [56] and
other beard dermal papilla cells [61]. The lack of effect when testosterone was
352 RANDALL
FIGURE 8 Beard dermal papilla cells produce significant amounts of 5 -

dihydrotestosterone, unlike pubic cells, mirroring hair growth in 5 -reductase deficient
patients. Dermal papilla cells from beard or pubic follicles were incubated for up to 24 hr
with 5 nM [3H] testosterone. The intracellular steroids were extracted and identified by
thin-layer chromatography and recrystallization [60]. Although beard cells rapidly formed
5 -dihydrotestosterone (a), pubic cells metabolized very little testosterone to 5 -
dihydrotestosterone even after 24hr (b). (Results from Ref. 60, diagram reproduced from
VA Randall, NA Hibberts, MJ Thornton, K Hamada, AE Merrick, S Kato, TJ Jenner, J De
Oliveria, AG Messenger. The hair follicle: a paradoxical androgen target organ. Horm
Res 54:243–250, 2000.)
added to media conditioned in the absence of testosterone confirming the

response was due to the production of a factor rather than a direct effect of
testosterone or its potentiation of a factor in beard condition media. In contrast,
when dermal papilla cells from balding scalp follicles both from men [63] and
from the stump-tailed macque [66] were investigated, testosterone inhibited the
growth-promoting effects of their media on keratinocytes. Again these in vitro
results reflect what would be expected from in vivo observations and support the
model. Nevertheless, it must be remembered that it is a hypothesis.
6
SUMMARY AND CONCLUSIONS
Androgens play a major role in altering the type of hair produced by human hair
follicles. The effect varies frotm stimulating a vellus follicle to enlarge enough to
produce a long, pigmented hair to the reverse transformation of terminal follicles
to vellus ones causing balding. The actual response is intrinsic to each individual
hair follicle and normally depends on its body site, presumably as a result of its
genetic programming during embryogenesis. Because of the important role of
human hair in social and sexual communication, both normal responses, such as
male pattern balding, and abnormal responses, such as hirsutism, may cause
marked psychological distress. These conditions are currently poorly controlled.
Our understanding of how androgens actually act in hair follicles has increased
recently, though the paradoxically different responses in different sites is still not
understood at a biochemical and molecular level. Androgen receptors have been
identified in follicular dermal papilla cells and the ability to metabolize
testosterone by cultured dermal papilla cells in vitro corresponds to hair growth
in 5 -reductase deficiency patients. These results suggest that metabolism of
testosterone, the main male circulating androgen, to 5 -dihydrotestosterone is
required for androgen action in most hair follicles, although not for axillary or
female pattern pubic hair growth. This understanding of the requirement for 5 -
reductase has led to the development of a 5 -reductase type 2 inhibitor,
finasteride, for the treatment of early stages of male pattern baldness. Finasteride
is also being tried for hirsutism under specific conditions.
These results have supported the current model in which androgens act via the
mesenchyme-derived dermal papilla to influence the cells of the hair follicle.
Once bound to androgen receptors in the dermal papilla cells, the hormone
should alter the production of paracrine factors produced by these cells to
regulate the other cells of the follicle. The current focus of much hair follicle
research is to identify such paracrine regulatory factors in the follicle. The
expression of genes for several factors is altered by androgens either in vivo or in
vitro. These include insulin-like growth factor 1 [56], stem cell factor [65], and
the protease nexin-1 [68]. Nexin-1, or glia-derived nexin-1, is a potent inhibitor
of serine proteases and therefore regulates cell growth and differentiation in a
number of tissues [69]. The “nexin-1 effect” is modulated by extracellular matrix
354 RANDALL
components such as a type IV collagen in other tissues [70]. Since the dermal
papilla contains a large amount of extracellular matrix that is related to the size
of the hair produced [51], and since dermal papilla cells produce these
extracellular matrix components [71], androgen-regulated alterations in protease
nexin1 production by dermal papilla cells could cause changes in extracellular
matrix components by the dermal papilla, hence the size of the hair produced.
Our understanding of how the hair follicle works in detail is still rather
superficial. However, with the advent of new cell and molecular biological
approaches, further studies, particularly into specific genes regulated by
androgens in hair follicles, could lead to the development of better treatments for
androgen-dependent disorders.
REFERENCES
1. Dry FW. The coat of the mouse (Mus musculus). J Genet 1926; 16:32–35.
2. Kligman AG. The human hair cycle. J Invest Dermatol 1959; 33:307–316.
3. Flux JEC. Colour change of mountain hares (Lepus timidus scoticus) in northeast
Scotland. Zoology 1970; 162:345–358.
4. Reynolds EL. The appearance of adult patterns of body hair in man. Ann N Y Acad
Sci 1951; 53:576–584.
5. Marshall WA, Tanner JM. Variations in the pattern of pubertal change in girls.
Arch Dis Child 1969; 44:291–303.
6. Marshall WA, Tanner JM. Variations in the pattern of pubertal changes in boys.
Arch Dis Child 1970; 45:13–23.
7. Paus R, Müller-Röver S, McKay I. Control of the hair follicle growth cycle. In:
Camacho FM, Randall VA, Price VH (eds.) Hair and Its Disorders: Biology,
Pathology and Management, Martin Dunitz, London 2000; 45: 83–94.
8. Ebling FG, Hale PA, Randall VA. Hormones and hair growth. In: Goldsmith LA,
(ed) Biochemistry and Physiology of the Skin, Clarendon Press, Oxford 1991; 45:
660–6906.
9. Hamilton JB. Patterned loss of hair in man; types and incidence. Ann N Y Acad
Sci 1951; 53:708–728.
10. Ebling FJG, Randall VA. Hormonal actions on hair follicles and associated glands.
In: Skerrow D, Skerrow CJ (eds). Methods in Skin Research, John Wiley & Sons,
London 1985; 53:297–327.
11. Randall VA. The use of human dermal papilla cells in hair growth studies. In:
Whiting D (ed) Dermatologic Clinics 14:4: Update on Hair Disorders, Saunders
WB, Philadelphia 1996; 53:585–594.
12. Randall VA, Hibberts NA, Thornton MJ, Hamada K, Merrick AE, Kato S, Jenner
TJ, De Oliveria I, Messenger AG. The hair follicle: a paradoxical androgen target
organ. Horm Res 2001; 54:243–250.
13. Philpott M. The roles of growth factors in hair follicles: investigations using
cultured hair follicles. In: Camacho FM, Randall VA, Price VH (eds). Hair and Its
Disorders: Biology, Pathology and Management, Martin Dunitz, London 2000; 54:
103–113.
14. Thornton MJ, Thomas DG, Jenner TJ, Brinklow BR, Loudon ASI, Randall VA.
Testosterone or IGF-1 stimulated hair growth in whole organ culture only in
androgen-dependent red deer hair follicles. In: Van Neste D, Randall VA. Hair
Research in the Next Millennium, Elsevier Science, BV, Amsterdam 1996; 54:311–
314.
15. Randall VA, Sundberg JP, Philpott MP. Animal and in vitro models for the study
of hair follicles. J Invest Dermatol 2003; in press.
16. Hamada K, Randall VA. Balding scalp dermal papilla cells secreta a soluble factor
(s) which delay the onset of anagen in mice in vivo. J Invest Dermatol 2003; in
press.
17. Kay RNB, Ryder ML. Coat growth in red deer (Cervus elaphus) exposed a day-
length cycle of six months duration. J Zool, Lond 1978; 185:505–510.
18. Milne JA, Loudon ASI, Sibbald AM, McNeilly AS. Effects of melatonin and a
dopamine agonist and antagonist on seasonal changes in voluntary intake,
reproductive activity, and plasma concentrations of prolactin and tri-iodothyronine
in red deer hinds. J Endocrinol 1990; 125: 241–249.
19. Martinet L, Allain D, Weiner C. Role of prolactin in the photoperiodic control of
moulting in the mink (Mustela vison). J Endocrinol 1984; 103:9–15.
20. Dicks P, Russel AJF, Lincoln GA. The role of prolactin in the reactivation of hair
follicles in relation to moulting in Cashmere goats. J Endocrinol 1995; 143:441–
448.
21. Orentreich N. Scalp hair replacement in men. In: Montagna W. Dobson, RL,
Advances in Biology of Skin, Hair Growth Pergamon Press, Oxford 1969; 143: 99–
108.
22. Randall VA, Ebling EJG. Seasonal changes in human hair growth. Br J Dermatol
1991; 124:146–151.
23. Courtois M, Loussouarn G, Howseau S et al. Periodicity in the growth and
shedding of hair. Br J Dermatol 1996; 134:47–54.
24. Reinberg A, Lagoguey M, Chauffourinier JM, Cesselin F. Circannual and circadian
rhythms in plasma testosterone in five healthy young Parisian males. Acta
Endocrinol 1975; 80:732–743.
25. Smals AGH, Kloppenberg PWC, Benrad THJ. Circannual cycle in plasma
testosterone levels in man. J Clin Endocrinol Metabol 1976; 42:979–982.
26. Bellastella A, Criscuoco T, Mango A, Perrone L, Sawisi AJ, Faggiano M.
Circannual rhythms of LH, FSH, testosterone, prolactin and cortisol during puberty.
Clin Endocrinol 1983; 19:453–459.
27. Goodhart CB. The evolutionary significance of human hair patterns and skin
colouring. Adv Sci 1960; 17:53–58.
28. Ebling FJG. The mythological evolution of nudity. J Hum Evolut 1985; 14: 33–41.
29. Hamilton JB. Age, sex and genetic factors in the regulation of hair growth in man:
a comparison of Caucasian and Japanese populations. In: Montagna W, Ellis RA
(eds). The Biology of Hair Growth, Academic Press, New York 1958; 14:399–433.
30. Hamilton JB. A secondary sexual character that develops in men but not in women
upon aging of an organ present in both sexes. Anat Record 1946; 94: 466–467.
31. Norwood OTT. Male pattern baldness. Classification and incidence. South Med J
1975; 68:1359–70.
32. Ludwig E. Classification of the types of androgenic alopecia (common baldness)
arising in the female sex. Br J Dermatol 1977; 97:249–56.
356 RANDALL
33. Venning VA, Dawber R. Patterned androgenic alopecia. J Am Acad Dermatol

1988; 18:1073–1078.
34. Randall VA. Physiology and pathophysiology of androgenetic alopecia. In: HG
Burger, Endocrinology (Eds) IJ Degroot, JL Jameson Section XIV Male
Reproduction Ed., WB Saunders, Philadelphia 2001; 18: 2257–2268.
35. Girman CJ, Rhodes T, Lilly FRW, Guo SS, Siervogel RM, Patrick DL, Chumlea
WC. Effects of self-perceived hair loss in a community sample of men.
Dermatology 1998; 197:223–229.
36. Winter JSD Faiman C. Pituitary-gonadal relations in male children and
adolescents. Paediatr Res 1972; 6:125–135.
37. Winter JSD, Faiman C. Pituitary-gonadal relations in female children and
adolescents. Paediatr Res 1973; 7:948–953.
38. Hamilton JB. Effect of castration in adolescent and young adult males upon further
changes in the proportions of bare and hairy scalp. J Clin Endocrinol Metabol
1960; 20:1309–1318.
39. Quigley CA. The androgen receptor: physiology and pathophysiology. In:
Nieschlag E, Behre HM, Testosterone: Action, Deficiency, Substitution, Springer-
Verlag, Berlin 1998; 20:33–106.
40. Ziller C. Pattern formation in neural crest derivatives. In: Van Neste D, Randall VA.
Hair Research for the Next Millennium, Elsevier Science, Amsterdam. 1996; 20:1–
5.
41. Orentreich N, Durr NP. Biology of scalp hair growth. Clin Plast Surg 1982; 9: 197–
205.
42. Hamilton JB. Male hormone stimulation is a prerequisite and an incitant in
common baldness. Amer J Anat 1942; 71:451–480.
43. Jeffcoate W. The treatment of women with hirsutism. Clin Endocrinol 1993; 39:
143–150.
44. Kaufman KD, Olsen EA, Whiting D, Savi R, De Villez R, Bergfeld W and the
Finasteride Male Pattern Hair Loss Study Group: Finasteride in the treatment of
men with androgenetic alopecia. J Am Acad Dermatol 1988; 39: 578–589.
45. Kligman AM. The comparative histopathology of male-pattern baldness and
senescent baldness. Clin Dermatol 1988; 64:108–118.
46. Randall VA. The role of 5a-reductase in health and disease. In: Sheppard M,
Stewart P. Hormones, enzymes and receptors, Baillières Clin Endocrinol Metabol,
1994; 8:405–431.
47. Wilson JD, Griffin JE, Russell DW. Steroid 5a-reductase 2 deficiency. Endocrinol
Rev 1993; 14:577–593.
48. Jahoda CAB, Reynolds AJ. Dermal-epidermal interactions; adult folliclederived
cell populations and hair growth. In: Whiting DA (ed). Dermatologic Clinics 14.
Update on Hair Disorders, WB Saunders, Philadelphia 1996; 14: 573–583.
49. Randall VA. Androgens and human hair growth. Clin Endocrinol 1994; 40: 439–
457.
50. Randall VA, Thornton MJ, Hamada K, Redfern CPF, Nutbrown M, Ebling FJG,
Messenger AG. Androgens and the hair follicle: cultured human dermal papilla
cells as a model system. Ann N Y Acad Sci 1991; 642:355–375.
51. Elliot K, Stephenson TJ, Messenger AG. Differences in hair follicle dermal papilla
volume are due to extracellular matrix volume and cell number: implications for
the control of hair follicle size and androgen responses. J Invest Dermatol 1999;
113:873–877.
52. Randall VA, Thornton MJ Messenger AG. Cultured dermal papilla cells from
androgen-dependent human follicles (e.g. beard) contain more androgen receptors
than those from non-balding areas. J Endocrinol 1992; 133: 141–147.
53. Hibberts NA, Howell AE, Randall VA. Dermal papilla cells from human balding
scalp hair follicles contain higher levels of androgen receptors than those from
nonbalding scalp. J Endocrinol 1998; 156:59–65.
54. Thornton MJ, Hibberts NA, Street T, Brinklow BR, Loudon AS, Randall VA.
Androgen receptors are only present in mesenchyme-derived dermal papilla cells
of red deer (Cervus elaphus) neck follicles when raised androgens induce a mane in
the breeding season. J Endocrinol 2001; 168:401–408.
55. Choudhry R, Hodgins MB, Van der Kwast TH, Brinkman AO, Boersma WJA.
Localisation of androgen receptors in human skin by immunohistochemistry:
implications for the hormonal regulation of hair growth, sebaceous glands and
sweat glands. J Endocrinol 1992; 133:467–475.
56. Itami S, Kurata S, Takayasu S. Androgen induction of follicular epithelial cell
growth is mediated via insulin-like growth factor I from dermal papilla cells.
Biochem Biophys Res Commun. 1995; 212:988–994.
57. Sawaya ME, Price VH. Different levels of 5a-reductase type I and II, aromatase
and androgen receptor in hair follicles of women and men with androgenetic
alopecia. J Invest Dermatol 1997; 109:296–301.
58. Itami S, Kurata S,Takayasu S. 5a-Reductase activity in cultured human dermal
papilla cells from beard compared with reticular dermal fibroblasts. J Invest
Dermatol 1990; 94:150–152.
59. Thornton MJ, Liang I, Hamada K, Messenger AG, Randall VA. Differences in
testosterone metabolism by beard and scalp hair follicle dermal papilla cells. Clin
Endocrinol 1993; 39:633–639.
60. Hamada K, Thornton MJ, Liang I, Messenger AG, Randall VA. Pubic and axillary
dermal papilla cells do not produce 5a-dihydrotestosterone in culture. J Invest
Dermatol 1996; 106:1017–1022.
61. Thornton MJ, Hamada K, Messenger AG, Randall VA. Beard, but not scalp, dermal
papilla cells secrete autocrine growth factors in response to testosterone in vitro. J
62. Limat A, Hunziker T, Waelti ER, Inaebrit SP, Wiesmann U, Brathen LR. Soluble
factors from human hair papilla cells and dermal fibroblasts dramatically increase
the clonal growth of outer root sheath cells. Arch Dermatol Res 1993; 285:205–
210.
63. Hibberts NA, Randall VA. Testosterone inhibits the capacity of cultured cells from
human balding scalp dermal papilla cells to produce keratinocyte mitogenic
factors. In: Van Neste DV, Randall VA (eds.) Hair Research for the Next
Millennium, Elsevier Science, Amsterdam 1996; 285:303–306.
64. Lachgar S, Moukadiri H, Jonca F et al. Vascular endothelial growth factor is an
autocrine growth factor for hair dermal papilla cells. J Invest Dermatol 1996; 106:
17–23.
65. Hibberts NA, Messenger AG, Randall VA. Dermal papilla cells derived from beard
hair follicles secrete more stem cell factor (SCF) in culture than scalp cells or
dermal fibroblasts. Biochem Biophys Res Commun. 1996; 222: 401–405.
358 RANDALL
66. Obana N, Chang C, Uno H. Inhibition of hair growth by testosterone in the

presence of dermal papilla cells from the frontal bald scalp of the post-pubertal
stump-tailed macque. Endocrinology 1997; 138:356–361.
67. Blume-Peytavi U, Mandt N. Signalling molecules in human hair follicle cell
populations. In: Camacho FM, Randall VA, Price VH, Hair and Its Disorders:
Biology, Pathology and Management, Martin Dunitz, London 2000; 138: 95–101.
68. Sonada T, Asada Y, Kurata S, Takayasu S. The mRNA for protease nexin-1 is
expressed in human dermal papilla cells and its level is affected by androgen. J
69. Low DA, Scott RW, Baker JB, Cunningham DD. Cells regulate their mitogenic
response to thrombin through release of protease nexin. Nature 1982; 298: 476–
478.
70. Donovan FM, Vaughan PJ, Cunningham DD. Regulation of protein nexin-1 target
protease specificity by collagen type IV. J Biol Chem 1994; 269: 17199–205.
71. Messenger AG, Elliott K, Temple A, Randall VA. Expression of basement
membrane proteins and interstital collagens in dermal papillae of human hair
follicles. J Invest Dermatol 1991; 96:93–97.
15
Alopecia Areata: An Update on Etiology and
Pathogenesis
Andrew J.G.McDonagh, Rachid Tazi-Ahnini, and Andrew
G.Messenger
University of Sheffield, Sheffield, United Kingdom
Alopecia areata is a common chronic inflammatory disorder characterized by T-

cell infiltration of hair follicles and nails. The inheritance pattern is in keeping
with a polygenic inheritance model, and to date, three genetic susceptibility/
severity factors have been identified by means of a candidate gene approach. The
HLA DQB and DR alleles on chromosome 6p21.3 have been demonstrated to
confer a high risk of disease by both case–control and family-based studies.
Interleukin-1 cluster genes, mainly the interleukin-1 receptor antagonist on
chromosome 2q12.21, show a strong association with disease severity in alopecia
areata and several other autoimmune and inflammatory diseases. Our finding of
association of alopecia areata with MXI on chromosome 21q22.3 may explain the
increase of alopecia areata in Down’s syndrome. Taken together with the high
frequency of alopecia areata in the’ autoimmune polyglandular syndrome type I,
the autoimmune regulator (AIRE) gene on chromosome 21q22.3 is a strong
candidate gene in alopecia areata. Environmental factors may be involved in
initiating disease expression in alopecia areata, but their role remains
speculative. Research on pathogenesis has concentrated on the role of the
immune system and the nature of the hair follicle pathology, but understanding
of the relationship between these remains limited. The new genetic information
should help to stimulate research on disease mechanisms and new therapeutic
approaches.
1
EPIDEMIOLOGY, INHERITANCE, AND THE ROLE
OF INFECTION
Epidemiological information on alopecia areata is limited because only one
formal population study of the disorder has been published [1]. This work
examined the occurrence of alopecia areata in Olmsted County, Minnesota, from
1975 to 1989. The incidence rate was 0.1 to 0.2%, with an estimated lifetime risk
of 1.7%. There was no apparent sex difference in the incidence, and the number
of cases was insufficient to permit study of the distribution of age at onset. It was
estimated that around 8% of affected individuals developed chronic disease.
Substantial case collections of alopecia areata have been compiled in Europe, the
360 MCDONAGH ET AL.
United States, Japan, India, and Korea [2], but these have generally been drawn
from hospital clinic records, and no accurate indication of variation in disease
rates between populations is available. Most of the series have shown an
increased frequency of autoimmune diseases, especially thyroid disorders,
pernicious anemia, and vitiligo in probands, and this has been confirmed also for
family members [3, 4]. Type I diabetes mellitus, in contrast, is reduced in
frequency in probands with alopecia areata but significantly increased in their
relatives [3]. Several reports have described an association of alopecia areata
with atopic disorders, particularly atopic eczema, but this was not confirmed in a
large series from India [5].
Most cases of alopecia areata seem to be sporadic, and the reported frequency
of a positive family history varies widely between the series, from around 4% to
28% (Table 1). Many factors other than biological differences between
populations might explain this wide range, and it is notable that variation in the
frequency of a positive family history is greatest in the smaller series (Table 1).
In larger series with at least 200 cases, the frequency of a positive family history
ranges from 9 to 22%, and there is an interesting trend suggestive of possible
correlation with the proportion of female subjects in the series. The possibility
that females may have a higher frequency of affected relatives was also raised by
Friedmann [6], but it is unclear whether this reflects a true difference or merely
heightened awareness of alopecia in women. It is uncommon to encounter
families in which more than two members have alopecia areata, but as many as
seven affected members
TABLE 1 Frequency of a Family History in Alopecia Areata

Investigator(s) Region Number of Patients Family history (%)
Sabourauda France 500 20
Brownb United Kingdom 135 20
Andersonc United Kingdom 114 19
Muller & United States 736 10
Winkelmannd
Gip et al.e Sweden 269 17
Sauder et al.f United States 98 27
Friedmann [6] United Kingdom 151 28
Lutz & Bauerg Germany 167 6.6
De Waard-van der Holland 209 17
Spek et al.h
Gollnick and Germany 149 11.4
Orfanosi
van der Steen et al. Holland/Germany 348 16
[9]
Wang et al. [3] United States 517 19
Colombe et al. [8] United States 131 20
ALOPECIA AREATA 361
Investigator(s) Region Number of Patients Family history (%)

Roj Korea 905 11.5
Sharma et al.k India 808 9
a R Sabouraud. Sur I’étiologie de la pelade. Arch Dermato-Syphiligra Clin d’Hôpital St
Louis 1:31–49, 1929.

b WH Brown. The aetiology of alopecia areata and its relationship to vitiligo and possibly
sclerodermia. Br J Dermatol 41:229–323, 1929.

c I Anderson. Alopecia areata: a clinical study. Br Med J ii:1250–1252, 1950.
d SA Muller, RK Winkelmann. Alopecia areata. Arch Dermatol 88:290–297, 1963.
e L Gip, A Lodin, L Molin. Alopecia areata: a follow-up investigation of outpatient
material. Acta Dermatol Venereol (Stockh) 49:180–188, 1969.

f DN Sauder, WF Bergfeld, RS Krakauer. Alopecia areata; an inherited autoimmune
disease. In: Brown AC, Crounse AG, eds. Hair: Trace Elements and Human
Disease New York: Praeger, 1980, pp 343–347.
g G Lutz, R Bauer. Autoimmunity in alopecia areata. An assessment in 100 patients.
Hautarzt 39:5–11,1988.
h FB De Waard-van der Spek, AP Oranje, DM De Raeymaecker, JD PeereboomWynia.
Juvenile versus maturity-onset alopecia areata—a comparative retrospective

clinical study. Clin Exp Dermatol 14:429–433, 1989.
i H Gollnick, CE Orfanos. Alopecia areata: pathogenesis and clinical picture. In: Orfanos
CE, Happle R, eds. Hair and Hair Diseases. Berlin: Springer-Verlag, 1990, pp
529–569.
j Bl Ro. Alopecia areata in Korea (1982–1994). J Dermatol 22:858–864, 1995.
k VK Sharma, G Dawn, B Kumar. Profile of alopecia areata in northern India. Int J
Dermatol 35:22–27, 1996.
have occasionally been recorded [7]. Patches of alopecia areata may pass
unobserved at any age, and the first patch may not occur until old age, producing
particular difficulty with designation of family members as unaffected when
disease status is recorded early in life. Age at onset of disease in alopecia areata
has been relatively little studied, but the apparent age distribution has varied
markedly between series, with some showing a peak in the childhood years and
others as late as the fourth decade.
Colombe et al. [8] found that a family history was more common in
individuals developing alopecia areata before the age of 30 (37% vs 7.1 % in
cases with onset after 30). Groups of authors have aggregated cases differently
for analysis, and it is difficult from the literature to derive trends regarding the
distribution of age at onset. In a personal series of 450 cases, we have observed a
striking difference in the profile of age at onset of alopecia areata between atopic
and nonatopic individuals in whom the mean age at onset is many years later
(unpublished data).
The risk of alopecia areata in the children of a proband is around 10-fold
greater than that in the general population [9], but except in occasional families,
alopecia areata seems not to be inherited in a simple Mendelian fashion and the
genetic basis appears to be multifactorial. There have been several case reports
of alopecia areata in twins but only a single study looking at concordance rates in
362 MCDONAGH ET AL.
monozygotic and dizygotic pairs [10]. In this investigation, there was a

concordance rate of 55% for alopecia among monozygotic twins, with no
concordance among the dizygotic pairs included. However, the numbers studied
were insufficient to allow a precise estimate of the genetic contribution in
alopecia areata.
The idea that alopecia areata is due to infection, either directly or as a
consequence of a remote “focus of infection,” has a long history and still cannot
be ruled out. It was the predominant etiological theory until well into the
twentieth century, and sporadic reports connecting alopecia areata with infective
agents continue to appear. Skinner and colleagues [11] reported finding mRNA
for cytomegalovirus in alopecic lesions, but this was not confirmed in a
subsequent study from Italy [12]. There are occasional reports of epidemic
alopecia areata [13], though most date from the early years of the twentieth
century, and the clinical descriptions make it difficult to assess whether the
subjects truly had alopecia areata or some other type of patchy hair loss. There
are also a few reports of alopecia areata in husband and wife [14, 15], although
this may be coincidence. There are several reports of an apparent association
between alopecia areata and drugs, but no single drug or class of drugs
predominates, and again, these associations may be coincidental.
The “external” factor most frequently implicated in alopecia areata is
psychological stress. The significance of such an association is difficult to
establish because of the problems in performing a controlled investigation, and
published evidence is conflicting to the extent that no firm conclusion can be
reached. Despite the anecdotal nature of much of the evidence, it is possible that
environmental factors are responsible for triggering alopecia areata in some
patients. If so, it seems likely that a diversity of factors can operate in this way.
2
GENETIC ASSOCIATIONS
No genome-wide scan has been performed in alopecia areata, and the genetics is
still poorly understood. The clinical associations, particularly with other
autoimmune and inflammatory disorders, have pointed the way for candidate
gene analysis by means of case–control studies.
2.1
Major Histocompatibility Complex Genes
Study of the role of specific genes in alopecia areata came with the advent of
human leukocyte antigen (HLA) analysis, and several studies have now indicated
strong association between these antigens and the disorder. Analyses of antigens
in HLA class I failed to show consistent patterns of association, but firmer
associations have been established between alopecia areata and HLA class II genes
[2]. Most of the studies have shown an increased frequency of DR4, DR5
ALOPECIA AREATA 363
(DR11), and DQ3. Serological techniques originally suggested that DR4 and
DR5 were associated with severe forms of alopecia areata, and this was
subsequently confirmed by molecular typing. Colombe et al. found an increase in
the broad antigen DQ3 in all patients in their study, suggesting that this may act
as a susceptibility factor for alopecia areata [8]. The DQB1*0301 allele (a
subtype of DQ3, which is in linkage disequilibrium with DR5) was associated
with severe alopecia but not with ne newly diagnosed patchy disease. There was
also a strong association between alopecia totalis/universalis and the DR11 allele
DRB1*1104, which was absent in milder disease. The association with
DQB1*0301 had previously been reported by Morling et al. [16] and Welsh et
al. [17], who also showed an increase in the frequency of DQ3 that was greater in
alopecia totalis/universalis than in patchy alopecia. De Andrade et al. [18]
confirmed the importance of DQBl *03 alleles, which were present in 85 % of
alopecia areata patients compared with 46% of controls [odds ratio (OR) 7.1,
p<0.000001). In the only family-based study of HLA association and linkage in
alopecia areata, these authors reported that an association between alleles of
HLA-DQB1*0302, *0601, *0603 and HLA-R4, DR6 had been shown by the
transmission disequilibrium test [18]. Linkage analysis in 75 families supported
linkage between alopecia areata and HLA class II loci with maximal logarithm
of the odds scores of 2.42 for HLA-DQB at 5% recombination and 2.34 for HLA-
DR at 0% recombination.
2.2
Cytokine Genes
Interleukin 1 (IL-1) is a primary cytokine involved in mediating inflammatory
responses. The IL-1 gene cluster on chromosome 2 includes genes for the
proinflammatory IL-1 proteins, their cell membrane receptors, the
antiinflammatory IL-1 receptor antagonist, and its homologue, IL1F5 (IL1L1)
whose function is less clear. IL-1 may have a direct effect on hair growth: in hair
follicle organ cultures, it inhibits growth of the hair fiber and induces
morphological changes resembling those seen in alopecia areata [19, 20]. Tarlow
et al. [21] reported an association between the severity of alopecia areata and
inheritance of allele 2 of a five-allele variable-number tandem repeat
polymorphism in intron 2 of the interleukin-1 receptor antagonist gene (IL1RN).
We subsequently confirmed in 165 patients and 1150 controls that homozygosity
for the rare allele of IL1RN (IL1RN*2) was significantly associated with alopecia
areata [OR 1.89; 95% CI confidence interval (CI) 1.09, 3.28; p=0.02] and
demonstrated a novel association involving a polymorphism of the IL-1 receptor
antagonist homologue IL1F5 at position + 4734, IL1RN+2018, and alopecia areata
[22]. The effect of a genotype combining three copies of the rare alleles at the
ILlRN and IL1F5 loci conferred a more than additive increase in the risk of
disease compared to IL1RN+2018 or IL1F5+4734 alone (OR 3.37; 95% CI 1.60,
7.06; p=0.002), suggesting possible synergy between the ILlRN and IL1F5
364 MCDONAGH ET AL.
genes. This effect was stronger in patients with severe disease (alopecia totalis/
universalis) (OR 4.62; 95% CI 1.87, 11.40; p=0.0022) and in those with early
age at onset (< 20 years) (OR 6.38; 95% CI 2.64, 15.42; p=0.0002.) The findings
suggested that these polymorphisms within IL1RN and IL1F5 themselves or a
gene in linkage disequilibrium with IL1RN and IL1F5 predispose people to the
more severe forms of alopecia areata.
IL1RN variants are associated with the severity of several other inflammatory
autoimmune diseases, including ulcerative colitis, lichen sclerosus, psoriasis,
myasthenia gravis, multiple sclerosis, and rheumatoid disease.
In a recent family-based study, Barahamani et al. [23] failed to confirm from
131 trios the association of IL1RN genotypes with alopecia universalis.
However, in this study there were only 88 individuals with alopecia universalis,
including both probands and other affected family members. Moreover, since the
parents of the probands are unlikely all to be heterozygous for IL1RN variants,
the number of transmissions in this study may be insufficient for statistical
power to detect the difference between the transmitted and nontransmitted
IL1RN allele 2.
In a small case–control association study, Galbraith et al. [24] found evidence
of increased susceptibility for alopecia areata with certain IL-1/immunoglobulin
K light chain (Km) genotypes. In a larger study using the same IL1B marker, we
found no significant association of IL1B-511 or IL1B+3954 genotypes with the
overall data set, or with disease severity or age at onset [25]. The results
suggested the possibility of an association with IL1A+4845 in the overall data set
(OR 1.39; 95% CI 1.00, 1.93). This was due mainly to the contribution from mild
cases of alopecia areata (OR 1.48; 95% CI 0.96, 2.29), suggesting that IL-1 may
have a particular role in the pathogenesis of this subgroup.
Like IL-1, tumor necrosis factor a (TNF- ) has a potent inhibitory effect on hair
growth in vitro [20]. TNF- is encoded by a gene in the HLA class III region,
and a polymorphism of this gene has been shown to be strongly associated with
certain autoimmune/inflammatory diseases including systemic lupus
erythematosus (SLE), rheumatoid arthritis, dermatitis herpetiformis, and celiac
disease. TNF- polymorphisms were investigated in alopecia areata in a small
study on 50 cases by Galbraith and Pandey [26], who demonstrated a significant
difference inTNF- genotypes between patients with patchy disease and those
with alopecia totalis/universalis. However, there was no difference between
disease and control groups overall. The role of TNF- in the pathogenesis of
alopecia areata remains to be explored.
2.3
Chromosome 21
Alopecia areata is increased in Down’s syndrome with a frequency of up to 8.8%
[27]. Down’s syndrome results from an additional copy (full or partial) of
chromosome 21, and the Down’s syndrome region of chromosome 21 may
ALOPECIA AREATA 365
include genes involved in the pathogenesis. As with other autoimmune

conditions such as thyroid disorders and celiac disease that occur more
commonly in Down’s syndrome, there is a general question of whether the
mechanism involves disomic homozygosity for individual polymorphic genes,
increased dosage of nonpolymorphic genes, or a more complex set of gene
interactions.
Down’s syndrome cells show increased sensitivity to the effect of type I
interferons in vitro [28]. MX1 is the gene encoding the interferon-induced p78
protein MxA, which confers resistance to influenza viruses, and some years ago
we demonstrated that MxA protein is strongly expressed in lesional anagen hair
bulbs from patients with alopecia areata but not in normal follicles [29].
Screening of MX1 revealed four single nucleotide polymorphisms concentrated
within 147 base pairs in intron 6 and showing strong linkage disequilibrium [30].
In a case–control association study for the MX1 (+ 9959) polymorphism in 165
alopecia areata patients and 510 controls we found a significant association with
the condition (OR 1.79; 95% CI 1.21–2.66; p=0.0036). The risk of disease was
greater for patchy
TABLE 2 HLA Class II Antigens in Alopecia Areata

Investigator(s) Region Number of Association Relative Risk
Patients
Frentz et al.a United States 22 DR4 2.3
DR5 4.7
Friedmann [6] United 65 DR4 Not reported
Kingdom
Orecchia et al.b Italy 127 DR4 3.1
Odum et al.c Denmark 41 DR4 2.0
DPW4 5.1
Duvic et al.d United States 98 DR4 2.1
Zhang et al.e United 54 DR4 2.8
Kingdom
Morling et al. Denmark 20 DQB1*0301 6.1
[16] (DQ7)
Welsh et al. United States 85
[17]
All DQ3 4.2
AT/AU DQ3 12.1
Colombe et al. United States 131
[8]
All DQ3
AT/AU DRB1*1104 30.2
(DR11)
366 MCDONAGH ET AL.
Investigator(s) Region Number of Association Relative Risk

Patients
DQB1*0301
(DQ7)
De Andrade et United States 192 DQB1*0302,
al. [18] *0601, *0603
DR4, DR6
a G Frentz, K Thomsen, BK Jakobsen, A Svejgaard. HLA-DR4 in alopecia areata. J Am
Acad Dermatol 14:129–130, 1986.

b G Orecchia, MC Belvedere, M Martinetti, E Capelli, G Rabbiosi. Human leukocyte
antigen region involvement in the genetic predisposition to alopecia areata.

Dermatologica 175:10–14,1987.
c N Odum, N Morling, J Georgsen, BK Jakobsen, G Frentz, GF Jensen, L Fugger, A
Svejgaard. HLA-DP antigens in patients with alopecia areata. Tissue Antigens

35: 114–117,1990.
d M Duvic, MK Hordinsky, VC Fiedler, WR O’Brien, R Young, JD Reveille. HLA-D
locus associations in alopecia areata. DRw52a may confer disease resistance.

Arch Dermatol 127:64–68,1991.
e L Zhang, AP Weetman, PS Friedmann, DB Oliveira. HLA associations with alopecia
areata. Tissue Antigens. 38:89–91, 1991.
alopecia areata (mild disease) and with early age at onset (OR 2.34; 95% CI 1.24–
4.43; p=0.0072). This supports a role for chromosome 21 genes in the Down’s
syndrome region in the pathogenesis of alopecia areata. However, there is an
even stronger association with the autosomal recessive disorder autoimmune
polyglandular syndrome type I (APECED: autoimmune polyendocrinopathy-
candidiasis-ectodermal dystrophy), in which about 30% of sufferers have
alopecia areata [31]. These patients also suffer from autoimmune
hyperparathyroidism, thyroid disorders, vitiligo, and hypogonadism. The mutant
gene in APECED is known as the autoimmune regulator (AIRE) gene and maps
to chromosome 21q22.3. The AIRE gene is an activating element for a nuclear
transcription factor [32]. The expression profile of AIRE and the autoimmune
manifestations of APECED provide strong evidence of involvement of AIRE in
the immune system, but its role in the pathogenesis of alopecia areata is not yet
understood.
3
AUTOIMMUNITY
Autoimmunity as a major disease mechanism was first suggested by Rothman
following a paper presented by Van Scott [33]. In addition to associations with
autoimmune diseases, a variety of nonspecific abnormalities in peripheral T-cell
numbers and function have been reported (reviewed in Ref. [34]). Circulating
autoantibodies to hair follicle tissue have also been detected in patients with
alopecia areata [35]. Such antibodies also occur in normal subjects but less
ALOPECIA AREATA 367
frequently, and at lower titer. They recognize various epithelial compartments

within the hair follicle and appear to be targeted against intracellular antigens
[36]. Antibody binding has not been demonstrated in vivo in humans, and the
role of antibodies in the pathogenesis is unclear. Passive immunization with
alopecia areata serum failed to induce hair loss in human skin grafted onto nude
mice [37]. However, it has been reported that serum from a horse with an
alopecia areata-like form of hair loss caused local inhibition of hair growth when
injected into murine skin, whereas serum from a normal horse did not [38].
The best direct evidence implicating circulating immune factors in the
pathogenesis of alopecia areata comes from transplantation experiments carried
out by Gilhar and colleagues. First, they showed that hair growth recovered in
alopecic skin transplanted onto athymic nude mice [39]. In later experiments (in
which SCID animals rather than athymic nude mice were used as graft
recipients), alopecia was induced in grafted skin by the injection of autologous T
lymphocytes incubated with hair follicle extracts and antigen-presenting cells
[40]. Tcells not incubated with hair follicle extracts failed to cause hair loss.
Taken together with the T-cell depletion studies on the DEBR model (Sec. 5.1),
the results of these experiments strongly suggest that alopecia areata is a T-cell-
mediated disease. It is sometimes suggested that the therapeutic efficacy of
agents such as cyclosporin and tacrolimus (FK506) in alopecia areata supports the
role of autoimmunity in the pathogenesis, but these agents have many different
actions on the immune system as well as possible direct effects on the hair
follicle [41].
4
PATHOLOGY
4.1
Pathodynamics
Alopecia areata alters the dynamics of hair growth. This is important, first
because it causes difficulty in interpreting the histopathology when one is
attempting to distinguish disease-related features from changes occurring
normally during the hair growth cycle, and second because understanding the
pathodynamic changes can provide clues to the nature of the hair follicle
pathology.
The most detailed study of early-stage changes in alopecia areata was carried
out by Eckert and colleagues [42]. They determined anagen/telogen ratios in
hairs plucked from demarcated concentric zones around the periphery of
expanding bald patches. Hair loss was preceded by a large increase in the
proportion of telogen hairs and an increase in the proportion of hairs showing
dystrophic features. They investigators concluded that the initial event in
alopecia areata is precipitation of anagen follicles into telogen. Less severely
368 MCDONAGH ET AL.
affected follicles may remain in anagen for a time, but they produce a dystrophic
hair and eventually also undergo telogen conversion. In keeping with these
observations, biopsy samples from the margins of expanding lesions of alopecia
areata show most follicles in catagen or early telogen [43]. It is not clear whether
follicles attain telogen via normal catagen. Exclamation point hairs may have a
well-formed club root identical to that of a normal telogen hair. However, the
root is frequently narrowed, and club hairs fall out more readily than normal,
suggesting that anchoring of the hair within the follicle is defective. Anagen
follicles in this site usually show peribulbar inflammation, although it may be
necessary to take the biopsy sample peripheral to the patch of hair loss to
demonstrate such follicles.
What happens next? Van Scott [33] studied biopsy samples from patches of
alopecia areata and found an average of 58% of follicles in anagen, suggesting
that reentry into anagen takes place. In early lesions there was a reduction in the
size of the lower follicle with preservation of the upper part of the follicle and
the sebaceous gland. In long-standing disease the entire follicle became smaller.
The matrix of these miniaturized anagen follicles was mitotically active and
produced a normal inner root sheath, but the cortex was incompletely keratinized.
Van Scott interpreted these changes as indicating arrest of follicle development
in anagen IV [33]. Our own findings have been in broad agreement with those of
Van Scott. Using horizontal sectioning of biopsy samples from alopecia totalis/
universalis and from the center of bald patches, we found that anagen follicles
failed to develop beyond anagen III/IV. At this stage the inner root sheath is a
conical keratinized structure, and the hair cortex has just started to differentiate
beneath it. We suggested that follicles return prematurely to telogen from anagen
III/IV and undergo repeated truncated cycles. As disease activity subsides,
follicles are able to progress further into anagen [43].
Except in very long-standing alopecia areata, hair follicles are retained even in
clinically hairless scalp. When alopecia areata has persisted for many years,
particularly in the universal form, there may be a decline in follicle density,
possibly associated with fibrosis of the perifollicular connective tissues.
4.2
Immunopathology
A perifollicular and intrafollicular inflammatory cell infiltrate is characteristic of
alopecia areata. This is most striking early in the disease, when anagen hair bulbs
appear to be affected preferentially. In established bald patches, the
inflammatory infiltrate is often sparse, but immunohistology will usually reveal
lymphocytes within the dermal papilla and matrix epithelium of anagen follicles.
In contrast to the inflammatory scarring alopecias, little or none of the
inflammatory infiltrate is seen around the isthmus of the hair follicle, the
proposed site for hair follicle stem cells [44]. This may explain why follicles are
not destroyed in alopecia areata. The inflammatory infiltrate is composed mainly
ALOPECIA AREATA 369
of activated T lymphocytes, with a preponderance of CD4 cells, and an

admixture of macrophages and Langerhans cells [45, 46]. In lesional anagen
follicles, lymphocytic infiltration of the dermal papilla and bulbar epithelium
may be accompanied by increased expression of HLA class I [47, 52] and class
II antigens [48] and of intercellular adhesion molecule 1 (ICAM-1) [49, 50]. This
is a common feature in inflammatory diseases characterized by lymphocytic
infiltration and is thought to be secondary to the local release of T-cell cytokines.
4.3
Hair Follicle Pathology
Cells of several different types and differentiation pathways are found in the hair
bulb, but which of these is the focus of the pathology is unknown. Based on the
following observations, we suggested that matrix epithelium undergoing early
cortical differentiation is the primary target of an immune attack on the hair
follicle. First, these cells show vacuolar degeneration in lesional anagen follicles
[51, 52]. This explains the formation of the exclamation point hair as it leads to a
focal zone of weakness in the hair shaft, which then breaks on reaching the skin
surface. Second, the pathodynamic changes can be explained on this basis: the
follicle is able to protect itself by reverting to telogen, in which cortical
differentiation does not occur. The follicle reenters anagen normally but is
restrained from developing beyond the stage when cortical differentiation
commences (anagen III/IV). Finally, the precortical region is the preferential site
of aberrant expression for major histocompatibility complex classes I and II.
On the other hand, it is possible that pathological changes in the precortical
matrix are secondary to dysfunction of the dermal papilla. This was first
suggested by the work of Van Scott and Ekel [53], who demonstrated alterations
in the morphometric relationships between the dermal papilla and the hair bulb
matrix in alopecia areata. More recently, ultrastructural abnormalities in cellular
morphology within the dermal papilla have been described in hair follicles from
both lesional and clinically nonlesional sites [54]. Whether such abnormalities of
clinically nonlesional tissue are indicative of a disease-prone phenotype or
merely represent low-grade disease involvement is not clear.
The sparing of white hair sometimes seen has led to suggestions that alopecia
areata is primarily a disease of hair bulb melanocytes [55]. Alopecia areata
shows other pigmentary features, including reduced pigmentation in regrowing
hairs and an association with vitiligo. However, the melanocyte hypothesis does
not explain why sparing of white hair is often a relative phenomenon and is
sometimes absent.
370 MCDONAGH ET AL.
5
ANIMAL MODELS
5.1
Dundee Experimental Bald Rat (DEBR)
The DEBR strain of the brown hooded rat arose as a spontaneous mutation at the
University of Dundee, Scotland [56]. Animals grow a normal first coat of hair but
then become progressively hairless, and skin histology confirms the persistence
of hair follicles, mostly in a dystrophic anagen state. Perifollicular and
intrafollicular lymphocytic infiltration is a prominent feature, and vacuolar
degeneration occurs in the cortex of some lesional anagen follicles. Increased
expression of HLA class I and II molecules in the dermal papilla and precortical
matrix is seen in a pattern similar to human alopecia areata [57]. Hair regrowth in
DEBR alopecia can be stimulated by photochemotherapy (i.e., psoralen with
ultraviolet A irradiation), topical minoxidil, systemic cyclosporin A [58], and
topical tacrolimus [59]. Partial regrowth can also be induced using monoclonal
antibodies to deplete circulating CD4 or CD8 cells, suggesting that T cells play
an active role in the pathogenesis [60].
5.2
C3H/HeJ Mouse
A diffuse nonscarring alopecia with clinical and pathological features similar to
alopecia areata was reported by Sundberg et al. [61] in a large production colony
of C3H/HeJ mice. On the dorsal skin, the alopecia developed in circular areas
with disease involvement restricted to anagen follicles. Pedigree analysis
suggested that the disease was inherited. Alopecia was commoner in aging
animals, and the frequency was highest in mice selectively bred for inflammatory
bowel disease. Subsequent studies have revealed considerable similarity in
histopathology, immunological features, and response to therapeutic agents
between C3H/HeJ alopecia and human alopecia areata. The possible role of steroid
hormonal factors has been examined in this model, with results suggesting
dihydroteststerone can confer resistance to hair loss while estradiol promotes
alopecia [62]. However, the relationship of these findings to human alopecia
areata is problematic.
6
CONCLUSIONS
There is now a considerable array of evidence for alopecia areata as a polygenic
autoimmune disease, but because of its diverse associations and clinical
presentations, it has been proposed that alopecia areata is a heterogeneous group
of diseases and not a single entity. At present, it is impossible to determine
ALOPECIA AREATA 371
whether this idea is correct. However, the evidence suggests that alopecia areata
is a multifactorial reaction pattern resulting from combinations of genetic and,
possibly, environmental factors. A large number of genes may be involved, and
their relative contributions will inevitably differ from person to person. Genes
controlling immune responses, inflammatory responses, and interactions between
the hair follicle and the immune system, as well as genes involved in regulating
the hair cycle, may be implicated. Each of these factors plays a role but none, on
its own, is sufficient to cause the disease.
Arguments over whether alopecia areata is primarily a disorder of the immune
system or the hair follicle may be impossible to resolve because alterations in
both immune function and hair follicle physiology may be necessary to cause
clinical expression of the disease. The concept that the hair follicle is an
immunologically privileged tissue [63], not normally subject to immune
surveillance, may provide the basis for a unifying hypothesis. A failure of
follicular immune privilege, which might occur for a variety of reasons, both
intra- and extrafollicular, would lead to an autoreactive attack on the hair
follicle.
REFERENCES
1. Safavi KH, Muller SA, Suman VJ, Moshell AN, Melton LJ, III. Incidence of
alopecia areata in Olmsted County, Minnesota, 1975 through 1989. Mayo Clin Proc
1995; 70:628–633.
2. Messenger AG, McDonagh AJG. Alopecia areata: aetiology and pathogenesis. In:
Camacho FM , Randall VA, Price VH editors. Hair and Its Disorders, London:
Martin Dunitz, 2000:177–186.
3. Wang SJ, Shohat T, Vadheim C, Shellow W, Edwards J, Rotter JI. Increased risk
for type I (insulin-dependent diabetes in relatives of patients with alopecia areata
(AA). Am J Med Genet 1994; 51:234–239.
4. Duvic M, Nelson A, de Andrade M. The genetics of alopecia areata. Clin Dermatol
2001; 19:135–139.
5. Sharma VK, Muralidhar S, Kumar B. Reappraisal of Ikeda’s classification of
alopecia areata: analysis of 356 cases from Chandigarh, India. J Dermatol 1998; 25:
108–111.
6. Friedmann PS. Clinical and immunologic associations of alopecia areata. Semin
Dermatol 1985;4:9–15.
7. Green J, Sinclair R. Seven members of a non-consanguineous Italian-Austra-lian
family with alopecia areata. Exp Dermatol 1999; 8:A438.
8. Colombe BW, Price VH, Khoury EL, Garovoy MR, Lou CD. HLA class II antigen
associations help to define two types of alopecia areata. J Am Acad Dermatol
1995; 33:757–764.
9. van der Steen P, Traupe H, Happle R, Boezeman J, Strater R, Hamm H. The
genetic risk for alopecia areata in first degree relatives of severely affected patients.
An estimate. Acta Dermatol Venereol (Stockh) 1992; 72:373–375.
372 MCDONAGH ET AL.
10. Jackow C, Puffer N, Hordinsky M, Nelson J, Tarrand J, Duvic M. Alopecia areata

and cytomegalovirus infection in twins: genes versus environment? J Am Acad
Dermatol 1998; 38:418–425.
11. Skinner RB J, Light WH, Bale GF, Rosenberg EW, Leonardi C. Alopecia areata
and presence of cytomegalovirus DNA. JAMA 1995; 273:1419–1420.
12. Tosti A, La Placa M, Placucci F, Gentilomi G, Venturoli S, Zerbini M, Musiani M.
No correlation between cytomegalovirus and alopecia areata. J Invest Dermatol
1996; 107:443.
13. Williams N, Riegert AL. Epidemic alopecia areata. An outbreak in an industrial
setting. J Occup Med 1971; 13:535–542.
14. Swift S. Folie a deux? Simultaneous alopecia areata in a husband and wife. Arch
Dermatol 1961; 84:94–96.
15. Zalka AD, Byarlay JA, Goldsmith LA. Alopecia a deux: simultaneous occurrence
of alopecia in a husband and wife. Arch Dermatol 1994; 130:390–392.
16. Morling N, Frentz G, Fugger L, Georgsen J, Jakobsen B, Odum N, Svejgaard A.
DNA polymorphism of HLA class II genes in alopecia areata. Dis Markers 1991; 9:
35–42.
17. Welsh EA, Clark HH, Epstein SZ, Reveille JD, Duvic M. Human leukocyte
antigen-DQBl*03 alleles are associated with alopecia areata. J Invest Dermatol
1994; 103:758–763.
18. de Andrade M, Jackow CM, Dahm N, Hordinsky M, Reveille JD, Duvic M.
Alopecia areata in families: association with the HLA locus. J Invest Dermatol
Symp Proc 1999; 43:220–223.
19. Harmon CS, Nevins TD. IL-1 alpha inhibits human hair follicle growth and hair
fiber production in whole-organ cultures. Lymphokine Cytokine Res 1993; 12:197–
203.
20. Philpott MP, Sanders DA, Bowen J, Kealey T. Effects of interleukins, colony-
stimulating factor and tumour necrosis factor on human hair follicle growth in vitro:
a possible role for interleukin-1 and tumour necrosis factor-alpha in alopecia
areata. Br J Dermatol 1996; 135: 942–948.
21. Tarlow JK, Clay FE, Cork MJ, Blakemore AI, Mcdonagh AJ, Messenger AG, Duff
GW. Severity of alopecia areata is associated with a polymorphism in the
interleukin-1 receptor antagonist gene. J Invest Dermatol 1994; 103: 387–390.
22. Tazi-Ahnini R, Cox A, McDonagh AJG, Messenger AG, di Giovine FS, Timms JM,
Dimitropolou P, Duff GW, Cork MJ. Genetic analysis of IL1RN and IL1L1 in
alopecia areata: strong association with severe disease and early age at onset. Eur J
Immunogenet 2002; 29:25–30.
23. Barahamani N, de Andrade M, Slusser J, Zhang Q, Duvic M. Interleukin-1 receptor
antagonist allele 2 and familial alopecia areata. J Invest Dermatol 2002; 118:335–
337.
24. Galbraith GM, Palesch Y, Gore EA, Pandey JP. Contribution of interleukin 1 ß and
KM loci to alopecia areata. Hum Hered 1999; 49:85–89.
25. Tazi-Ahnini R, McDonagh AJG, Cox A, Messenger AG, Britton JER, Ward SJ et al.
Association analysis of IL1A and IL1B variants in alopecia areata. Heredity 2001;
87:215–219.
26. Galbraith GM, Pandey JP. Tumor necrosis factor alpha (TNF-a) gene
polymorphism in alopecia areata. Hum Genet 1995; 96:433–436.
ALOPECIA AREATA 373
27. du Vivier A, Munro DD. Alopecia areata, autoimmunity and Down’s syndrome. Br
Med J 1975; i: 191–192.
28. Tan YH, Schneider EL, Tischfield J, Epstein CJ, Ruddle FH. Human chromosome
21 dosage: effect on the expression of the interferon induced antiviral state. Science
1974; 186:61–63.
29. McDonagh AJG, Elliott KR, Messenger AG. Mx protein: a new marker of type I
interferon activity in the skin. Br J Dermatol 1994; 132:648.
30. Tazi-Ahnini R, di Giovine FS, McDonagh AJG, Messenger AG, Amadou C, Cox A,
Duff GW, Cork MJ. Structure and polymorphism of the human gene for the
interferon-induced p78 protein (MXl): evidence of association with alopecia areata
in the Down syndrome region. Hum Genet 2000; 106: 639–645.
31. Betterle C, Greggio NA, Volpato M. Autoimmune polyglandular syndrome type 1.
J Clin Endocrinol Metab 1998; 83:1049–1055.
32. Kumar PG, Laloraya M, Wang CY, Ruan QG, Davoodi-Semiromi A, Kao KJ, She
JX. The autoimmune regulator (AIRE) is a DNA-binding protein. J Biol Chem
2001; 276:41357–41364.
33. Van Scott EJ. Morphologic changes in pilosebaceous units and anagen hairs in
alopecia areata. J Invest Dermatol 1958; 31:35–43.
34. McDonagh AJG, Messenger AG. The pathogenesis of alopecia areata. Dermatol
Clin 1996; 14:661–670.
35. Tobin DJ, Orentreich N, Fenton DA, Bystryn JC. Antibodies to hair follicles in
36. Tobin DJ, Hann SK, Song MS, Bystryn JC. Hair follicle structures targeted by
antibodies in patients with alopecia areata. Arch Dermatol 1997; 133:57–61.
37. Gilhar A, Pillar T, Assay B, David M. Failure of passive transfer of serum from
patients with alopecia areata and alopecia universalis to inhibit hair growth in
transplants of human scalp skin grafted on to nude mice. Br J Dermatol 1992; 126:
166–171.
38. Tobin DJ, Alhaidari Z, OlivryT. Equine alopecia areata autoantibodies target
multiple hair follicle antigens and may alter hair growth. A preliminary study. Exp
Dermatol 1998; 7:289–297.
39. Gilhar A, Krueger GG. Hair growth in scalp grafts from patients with alopecia
areata and alopecia universalis grafted onto nude mice. Arch Dermatol 1987; 123:
44–50.
40. Gilhar A, Ullmann Y, Berkutzki T, Assy B, Kalish RS. Autoimmune hair loss
(alopecia areata transferred by T lymphocytes to human scalp explants on SCID
mice). J Clin Invest 1998; 101:62–67.
41. Taylor M, Ashcroft AT, Messenger AG. Cyclosporin A prolongs human hair
growth in vitro.. J Invest Dermatol 1993; 100:237–239.
42. Eckert J, Church RE, Ebling FJ. The pathogenesis of alopecia areata. Br J
Dermatol 1968; 80:203–210.
43. Messenger AG, Slater DN, Bleehen SS. Alopecia areata: alterations in the hair
growth cycle and correlation with the follicular pathology. Br J Dermatol 1986;
114:337–347.
44. Cotsarelis G, Sun TT, Lavker RM. Label-retaining cells reside in the bulge area of
pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin
carcinogenesis. Cell 1990; 61:1329–1337.
374 MCDONAGH ET AL.
45. Perret C, Wiesner-Menzel L, Happle R. Immunohistochemical analysis of T-cell

subsets in the peribulbar and intrabulbar infiltrates of alopecia areata. Acta
Dermatol Venereol (Stockh) 1984; 64:26–30.
46. Wiesner-Menzel L, Happle R. Intrabulbar and peribulbar accumulation of dendritic
OKT 6-positive cells in alopecia areata. Arch Dermatol Res 1984; 276:333–334.
47. Bröcker EB, Echternacht-Happle K, Hamm H, Happle R. Abnormal expression of
class I and class II major histocompatibility antigens in alopecia areata: modulation
by topical immunotherapy. J Invest Dermatol 1987; 88:564–568.
48. Messenger AG, Bleehen SS. Expression of HLA-DR by anagen hair follicles in
49. Gupta AK, Ellis CN, Cooper KD, Nickoloff BJ, Ho VC, Chan LS, Hamilton TA,
Tellner DC, Griffiths CE, Voorhees. Oral cyclosporine for the treatment of alopecia
areata. A clinical and immunohistochemical analysis. J Am Acad Dermatol 1990;
22:242–250.
50. McDonagh AJG, Snowden JA, Stierle C, Elliott K, Messenger AG. HLA and
ICAM-1 expression in alopecia areata in vivo and in vitro: the role of cytokines. Br
J Dermatol 1993; 129:250–256.
51. Thies W. Vergleichende histologische Untersuchungen bei Alopecia areata und
Narbig-Atrophisierenden. Arch Klin Exp Dermatol 1966; 227:541–549.
52. Messenger AG, Bleehen SS. Alopecia areata: light and electron microscopic
pathology of the regrowing white hair. Br J Dermatol 1984; 110:155–162.
53. Van Scott EJ, Ekel TM. Geometric relationships between the matrix of the hair
bulb and its dermal papilla in normal and alopecic scalp. J Invest Dermatol 1958;
31:281–287.
54. MacDonald-Hull S, Nutbrown M, Pepall L, Thornton MJ, Randall VA, Cunliffe
WJ. Immunohistologic and ultrastructural comparison of the dermal papilla and
hair follicle bulb from “active” and “normal” areas of alopecia areata. J Invest
Dermatol 1991; 96:673–681.
55. Paus R, Slominski A, Czarnetzki BM. Is alopecia areata an autoimmuneresponse
against melanogenesis-related proteins, exposed by abnormal MHC class I
expression in the anagen hair bulb? Yale J Biol Med 1993; 66:541–544.
56. Michie HJ, Jahoda CA, Oliver RF, Johnson BE. The DEBR rat: an animal model of
human alopecia areata. Br J Dermatol 1991; 125:94–100.
57. Zhang JG, Oliver RF. Immunohistological study of the development of the cellular
infiltrate in the pelage follicles of the DEBR model for alopecia areata. Br J
Dermatol 1994; 130:405–414.
58. Oliver RF, Lowe JG. Oral cyclosporin A restores hair growth in the DEBR rat
model for alopecia areata. Clin Exp Dermatol 1995; 20:127–131.
59. McElwee KJ, Rushton DH, Trachy R, Oliver RF. Topical FK506: a potent
immunotherapy for alopecia areata? Studies using the Dundee experimental bald
rat model. Br J Dermatol 1997; 137:491–497.
60. McElwee KJ, Spiers EM, Oliver RF. In vivo depletion of CD8+ T cells restores
hair growth in the DEBR model for alopecia areata. Br J Dermatol 1996; 135: 211–
217.
61. Sundberg JP, Cordy WR, King LEJ. Alopecia areata in aging C3H/HeJ mice. J
ALOPECIA AREATA 375
62. McElwee KJ, Silva K, Beamer WG, King LE. Jr. Sundberg JP. Melanocyte and
gonad activity as potential severity modifying factors in C3H/HeJ mouse alopecia
areata. Exp Dermatol 2001; 10:420–429.
63. Westgate GE, Craggs RI, Gibson WT. Immune privilege in hair growth. J Invest
Dermatol 1991; 97:417–420.
16
The Structure and Properties of Nails and
Periungual Tissues
David de Berker
Bristol Royal Infirmary, Bristol, United Kingdom
Bo Forslind†
Except for reports on pathological conditions, nails have received little scientific
interest in the past in comparison with the structure and function of the
integument and its appendix, hair. This is remarkable in as much as the condition
and function of human fingernails receives a great deal of attention in everyday
life. This chapter deals mainly with structure-function relationships at the
macromolecular and cellular levels, to provide a basis for an understanding of
the “hardness” of the human fingernail, all facts providing an understanding of
the nail as an important tool. From this foundation, we go on to discuss
brittleness, cleavage of the nail plate, and other features, as well as changes
observed in connection with disease.
Albeit civilized man to a great extent uses a wide assortment of mechanical
tools, the biological function of the human fingernail is to be a tool for grasping
and manipulating objects. In addition, the cutting property of the sharp end of
the nail allows peeling of fruits and removal of undesired parts of foodstuff as
well as serving defensively. The nail plate intensifies sensory discrimination in
the handling of minute objects and to a certain extent protects the fingertips from
traumatic impacts. From a sociological point of view, nails can serve as signals of
social class: for example, long fingernails indicated that Chinese mandarins were
not obliged to do manual work. Lacquered fingernails also signal social status, a
tradition that, interestingly, has its origin in the red lacquered fingernails of half-
blood women in Central America who were able to pass for whites by using red
lacquer to conceal their pigmented nail crescents.
The topographical location and the function of nails implies that they are
equivalent to the claws of other mammals. But why then are nails so inefficient
compared with the claws of mammals such as cats? My personal opinion is that,
like some other embryonic traits such as the lack of a furry hair coat, leaving us
with a comparatively bald body, our claw development was arrested at an early
stage as a trade-off necessary to achieve an upright position and to develop our
cerebrum to allow for mental activity and logical thinking. The excellent studies
of Le Gros-Clark [1, 2] offer gain support for such an idea, especially when with
†Deceased.
378 DE BERKER AND FORSLIND
respect to comparisons of the structures of claws and nails at different levels in

the primate series. However, it must be pointed out that Le Gros-Clark proposes
another interpretation of the development of nails.
This chapter emphasizes fingernails. However, many of the facts presented
here undoubtedly are relevant for toenails as well.
1
FUNDAMENTALS
1.1
Gross Anatomy and Terminology
It is necessary to have some fixed definitions of the structures discussed in this
chapter (Figs. 1–3).
Nail plate (nail): durable keratinized structure on the dorsal distal segment
of the digit that continues growing throughout life.
Lateral nail folds: the cutaneous folded structures providing the lateral
borders to the nail.
Proximal nail fold (posterior nail fold): cutaneous folded structure
providing the visible proximal border of the nail, continuous with the
cuticle.
Cuticle (eponychium): the layer of epidermis extending from the
proximal nail fold and adhering to the dorsal aspect of the nail plate.
Nail matrix (nail root): traditionally, this can be split into three parts [3],
the dorsal matrix which is synonymous with the ventral aspect of the
proximal nail fold; the intermediate matrix (germinative matrix—the
source of nail), which is the epithelial structure starting at the point at
which the dorsal matrix folds back on itself to underlie the proximal nail;
and the ventral matrix, which is synonymous with the nail bed and starts at
the border of the lunula, where the intermediate matrix stops, and is limited
distally by the hyponychium.
Lunula (half-moon): the convex margin of the intermediate matrix seen
through the nail. It is paler than the adjacent nail bed. It is most commonly
visible on the thumbs and great toes. It may be concealed by the proximal
nail fold.
Nail bed (ventral matrix, sterile matrix): the vascular bed upon which
the nail rests, extending from the lunula to the hyponychium. This is the
major territory seen through the nail plate.
Onychocorneal band: the distal margin of the nail bed, which has a
contrasting hue in comparison with the rest of the nail bed. Normally, this
is a transverse band of 1 to 1.5 mm of a deeper pink (Caucasian) or brown
(Afro-Carribbean), followed by a relatively avascular pale band (Fig. 2).
NAILS AND PERIUNGUAL TISSUES 379
FIGURE 1 (top) Longitudinal section and (bottom) dorsal view of the digit,
illustrating the terms used for anatomy of the nail unit.
Its color, or presence, may vary with disease or with compression, which
influences the vascular supply.
Hyponychium (contains the solenhorn): the cutaneous margin
underlying free nail, bordered distally by the distal groove.
Distal groove (limiting furrow): a cutaneous ridge demarcating the
border between subungual structures and the finger pulp.
FIGURE 2 Onychocorneal band.
1.2
Embryology
1.2.1
Morphogenesis
8 to 12 Weeks. Individual digits are discernible from the eighth week of gestation
[3]. The first embryonic element of the nail unit is the nail anlage, present from 9
weeks. At 10 weeks a distinct region can be seen and is described as the primary
nail field. This almost overlies the tip of the terminal phalanx, with clear
proximal and lateral grooves in addition to a welldefined distal groove. The
prominence of this groove is partly due to the distal ridge, thrown up proximally,
accentuating the contour. The primary nail field grows proximally by a wedge of
germinative matrix cells extending back from the tip of the digit. These cells are
proximal to both the distal groove and ridge. The spatial relationship of these two
latter structures remains relatively constant as the former becomes the vestigial
distal groove and the latter the hyponychium.
13 to 17 Weeks to Birth. At 17 weeks, the nail plate covers most of the nail bed
and the distal ridge has flattened. From 20 weeks, the nail unit and finger grow in
tandem, with the nail plate abutting the distal ridge. This now becomes termed the
hyponychium. The nail bed epithelium no longer produces keratohyalin, with a
more parakeratotic appearance. By birth the nail plate extends to the distal
groove, which becomes progressively less prominent. The nail may curve over
the volar surface of the finger. It may also demonstrate koilonychia. This
deformity is normal in the very young and a function of the thinness of the nail
plate. It reverses with age.
NAILS AND PERIUNGUAL TISSUES
FIGURE 3 Histological section of longitudinal biopsy sample of toenail stained with hematoxylin and eosin (×10). See Figure 1A for
orientation.
381
1.2.2
Tissue Differentiation
Keratin synthesis can be identified in the nail unit from the earliest stages of its
differentiation [4]. In 12- and 13-week embryos, the nail-matrix anlage is a thin
epithelial wedge penetrating from the dorsal epidermis into the dermis. This
wedge is thought to represent the “ventral matrix primordium.” Keratin
represents about 80% of the intracellular structural protein of epithelial cells. It
belongs to the family of intermediate filaments. There are many different
keratins with varied structural properties and localization within animals. They
are divided into two groups, the first of which are “soft”, epithelial keratins
commonly found in the skin. The second consists of “hard”, trichocyte or hair/
nail keratins found in hair, nail, thymus, tooth primordia, and tongue.
By week 15, hard keratins are seen throughout the nail bed and matrix. This
could have significance for theories of nail embryogenesis and growth, since
there is debate over the contribution made by the nail bed to nail growth [3, 5–8].
However, at 22 weeks, the layer of hard keratin-positive cells remains very thin
in the nail bed, whereas it is considerably thickened in the matrix. In the adult
nail, there have been reports of both the presence [9] and absence [4, 10–12] of hard
keratins in the nail bed.
Histological observation at 13 and 14 weeks reveals parakeratotic cells just
distal to this nail plate primordium staining for disulfydryl groups. This contrasts
to adjacent epithelium, suggesting the start of nail plate differentiation. This early
differentiation represents matrix formation, and Merkel cells have been detected
in the matrix primordium of human fetuses between weeks 9 and 15 [13].
Merkel cells may play a role in the development of epidermal appendages and
are detectable using monoclonal antibodies specific to keratin 20. Their role in
ontogenesis would explain their disappearance from the nail matrix after week 22
[13]. However, this is not a universal finding: in one study, researchers identified
an abundance of Merkel cells in the matrix of young adult and cadaver nail
specimens [14].
At the 13 to 22-week stage there is coincident increase in the expression of
hard keratins and the development of keratohyalin granules.
By 25 weeks, most features of nail unit differentiation are complete. Changes
may still occur in the chemical constitution of the nail plate after this date. A
decrease in sulfur and aluminum and a rise in chlorine have been noted as
features of full-term newborns in comparison to the nail plate of premature
babies [15]. An elevated aluminum level may correspond to bone abnormalities
that lead to osteopenia.
1.3
The Morphology of the Nail Plate and Periunguium
In some sections of this chapter we will refer to the axes of the nail plate in terms
of the three dimensions between the dorsal and ventral aspects of the nail:
longitudinal (x), transverse (y), and vertical (z). The nail plate is derived from the
matrix and continually growing out of the proximal nail fold. The ventral nail
plate is formed by the distal section of the nail matrix, with dorsal nail as the
product of proximal matrix. The distal border of this matrix is defined as the
lunula, the white crescentlike, part of the proximal nail. The nail bed is defined
as distal to the lunula and proximal to the onychocorneal or onychodermal band
(Fig. 2). The nail bed, which contains a rich vascular capillary bed, is supported
by connective tissue, adherent to the periosteum of the underlying distal phalanx.
At light microscopic resolution the cells of the nail matrix are cuboidal,
standing on a basal latnina and progressing to a trapezoidal outline (Fig. 4). As
they differentiate into nail, unlike normal skin, they do not develop a granular
layer (Fig. 5). The soft connective tissue above the nail fold contains collagen
fibers with preferred orientation perpendicular to the finger axis (i.e. in the
direction of the y axis). This organization of the fibrous unit of the connective
tissue will counteract an upward movement of cells derived from the nail matrix.
Thus the cells of the dorsal nail plate undergo a flattening process during their
continuous movement out of the nail fold (Fig. 6A) . At the level of the distal
lunula edge, these cells are fully keratinized and thus not containing any nucleus
or fragments thereof. In comparison to the fully mature cells of the dorsal nail
plate, cells of the ventral nail plate do not undergo such an excessive flattening
process during their complete keratinization (Fig. 6B). At the level of the distal
margin of the lunula, however, one can see a gradient of change in these cells
going from the matrix cells and moving upward (in the y-axis direction), toward
the cells connected to the lowest cells of the dorsal nail plate (Fig. 4).
The dynamic aspects of this growth process have been beautifully revealed in
an autoradiographic study by Zaias and Alvarez [7]. In this study, the position of
the autoradiographic grains allows the interpretation that the nail cells are
subjected to shear forces during growth. On the assumption that the same rate of
cell division occurs all over the nail matrix, the cells of the dorsal nail plate are
the ones subjected to the most effective shear forces, which cause these cells to
flatten out extensively. Therefore the dorsal nail plate appears to be grow faster
than the deepest part of the ventral plate. The assumption that the proximal
matrix derives nail at the same rate as the distal matrix is open to question. In
studies using immunohistochemical markers of cell proliferation, such as
proliferating cell nuclear antigen or Ki67, the proximal 50% of matrix is seen to
contribute 80% of the prolifera-tive fraction of matrix cells [16] (Fig. 7A). The
relative inactivity of the nail bed is well illustrated by the reduction of labeled
cells in this zone in contrast to the matrix (Fig. 7B). Cells deeper down in the
nail are subject to lesser shear forces and consequently are not deformed
(flattened) as much; hence their relative movement will be slower. This study was
supported by findings presented by Norton [17]. The influence of shear forces on
the final form of the nail cells was corroborated by an innovative transplantation
experiment of nail matrix to a skin site [18]. The nail matrix cell progenies grew
upward, as a stack of more or less cuboidal cells. We shall return to these
phenomena later.
Details of this interpretation of morphology are confirmed at electron
microscopic resolution. The cells of the dorsal nail plate are shown to be
represented in the longitudinal cross section (along the x axis) as lamellar cells with
a comparatively straight cell border. The cells are joined by means of an
intercellular dense substance that leaves no open intercellular spaces. In contrast,
the cells of the ventral nail plate show an undulating cell border and the
intercellular cement substance is discontinuous, leaving partially open
intercellular spaces. The height of these cells in cross section is several times
that of the cells in the dorsal nail plate. The overall electron density, however,
seems to be the same as in the dorsal nail plate. This corresponds to findings of
microradiographic studies [19]. The development and morphology of human
nails have been thoroughly studied at electron microscopic resolution by
Hashimoto [6, 20] and lately by Kitahara [21].
FIGURE 4 Distal matrix keratinocytes at an oblique angle to the axis of the overlying nail (a 350 × magnification of matrix sample is shown
later, in Fig. 10B). These keratinocytes will be shed to comprise nail.
385
FIGURE 5 Histological section of longitudinal biopsy sample of toenail stained with hematoxylin and eosin (× 200). At the proximal matrix,
the lower epithelial surface generates nail and has no granular layer. The upper epithelial surface represents the ventral aspect of the proximal
nail fold and develops a granular layer.
FIGURE 6 Vertical histological sections of nail plate with periodic acid-Schiff stain (× 400). Onychocytes in the dorsal nail plate (A) are
flattened in comparison to those of the ventral nail (B).
387
FIGURE 6 Continued.
FIGURE 7A Histological section of nail matrix (× 80), labeled with antibody to proliferating cell nuclear antigen: proximal matrix and nail
fold.
389
FIGURE 7B Histological section of nail matrix (× 80), labeled with antibody to proliferating cell nuclear antigen: distal matrix and nail bed. A
dense compartment of proliferating cells in the matrix (A) wanes with the transition between distal matrix and nail bed (B).
1.4
Immunohistochemistry of Nail Keratins and Other
Proteins
1.4.1
Keratins
The most extensive immunohistological investigations of the nail unit have
utilized keratin antibodies. The nail plate [10, 22], human embryonic nail unit
[4, 10, 23], accessory digit nail unit [24], and adult nail unit [23, 25, 26] have all
been examined. Using monospecific antibodies, de Berker et al. [24] detected
keratins 1 and 10 in a suprabasal location in the matrix and noted their absence
from the nail bed. Keratins 1 and 10 are “soft” epithe-lial keratins found
suprabasally in normal skin [27] and characteristic of cornification with terminal
keratinocyte differentiation. Their absence from normal nail bed is reversed in
disease, where nail bed corniflcaton is often seen, alongside development of a
granular layer and expression of keratins 1 and 10 (Fig. 8) [28]. The
development of a granular layer in subungual tissues, which can be interpreted as
a pathological sign in nail histology, is seen in a range of diseases and probably
is associated with changes in keratin expression [29]. With the development of
terminal differentiation in nail bed epithelium, adherence between nail bed and
overlying nail is reduced and sometimes lost. This produces the clinical feature of
onycholysis—a gap between the nail bed and nail (Fig. 9)
The “hard” keratin Ha-1 is found in the matrix. Keratin 7 has been found at
other sites in the nail unit and hair follicle, whereas Ha-1, detected by the
monoclonal antikeratin antibody LH TRIC 1, is limited to the matrix of the nail
(Fig. 10) and the germinal matrix of the hair follicle [11, 12]. The localization of
Ha-1 is a useful indicator of matrix location and histological organization. In
onychogryphosis, in which the nail is thickened, distorted, and grows slowly, the
matrix changes shape and position in relation to the proximal nail fold (Fig. 11).
At a histological level, the organization of matrix cells producing Ha-1 changes
as these cells become less ordered (Fig. 12). This sequence could be relevant to
the altered character of the substance of the nail in this condition.
Keratin 19 probably is not present in the adult matrix [4, 24, 26]. However,
Moll et al. [4] did detect keratin 19 at this site in 15-week embryo nail units.
Keratin 19 is also found in the outer root sheath of the hair follicle and lingual
papilla [10].
FIGURE 8 Histological sections of nail bed (×100): (A) hematoxylin and eosin stain and (B) monoclonal antibody to epithelial keratin, k10.
The nail bed in psoriasis changes differentiation, with development of a granular layer that can be seen to closely match expression of k10, a
keratin associated with terminal keratinocyte differentiation.
FIGURE 8 Continued.
FIGURE 9 Histological section of longitudinal biopsy sample of nail bed in the inflammatory disease lichen planus. The granular layer in the
nail bed (nb) is a sign of disease. This is associated with hyperkeratosis (hk) and onycholysis (o) and lifting of the nail plate (np).
The colocalization of hard and soft keratins in single cells of the matrix has
been observed by several workers in bovine hoof [21] and in human nail [25, 30, 31]
, suggesting that these cells are contributing both forms of keratin to the nail
plate. This dual differentiation continues in the in vitro culture of bovine hoof
matrix cells [30]. Culture of human nail matrix confirms the persistence of hard
keratin expression [32, 33].
Markers for keratins 8 and 20 are thought to be specific to Merkel cells in the
epidermis. Positive immunostaining for these keratins has been noted by Lacour
et al. [23] in adult nail matrix and de Berker et al. [11] in infant accessory digits.
Some workers have failed to detect Merkel cells, and while it seems likely that
they are present in fetal and young adult matrices, it may be that the cells are less
common or absent as people age [34].
The nail bed appears to have a distinct identity with respect to keratin
expression. Keratins 6, 16, and, to a lesser degree, 17 are all found in the nail bed
and are largely absent from the matrix [24]. This finding has gained clinical
significance with the characterization of the underlying fault in some variants of
pachyonychia congenita, where abnormalities of nail bed keratin lead to a
grossly thickened nail plate. Mutations in the gene for keratin 17 have been
reported in a large Scottish kindred with the PC-2, or Jackson-Lawlor, phenotype
[35, 36]. There is a crossover with steatocystoma multiplex, where the same
mutation of keratin 17 may cause this phenotype, which appears to be
independent of the specific keratin 17 mutation [36–39]. Mutations in the gene
coding for K6b produce a phenotype seen with K17 gene mutations [40].
Mutations in the K6a [41] and K16 [36] genes have been reported in PC-1,
originally described as the Jadassohn-Lewandsky variant of pachyonychia
congenita. Expression of keratins 6, 16, and 17 extend beyond the nail bed onto
the digit pulp and are thought to match the physical characteristics of this skin,
which is adapted to high degrees of physical stress [42]. In particular, expression
of keratin 17 is found at the base of epidermal ridges, which might also support
the idea that this keratin is associated with stem cell function.
1.4.2
Non-Keratin Immunohistochemistry
Haneke [26] has provided a review of other important immunohistochemically
detectable antigens. Involucrin is a protein necessary for the formation of the
cellular envelope in keratinizing epithelia. It is strongly positive in the upper two
thirds of the matrix and elsewhere in the nail unit [43] and weakly detected in the
suprabasal layers. Pancornulin and sciellin are also detected in the matrix [43].
The antibody HHF35 is considered to be specific to actin. It has been found to
show a strong membranous staining and weak cytoplasmic staining of matrix
cells [26].
FIGURE 10 Histological section of toenail matrix (×150), labeled with monoclonal antibody to Tric-1, specific to the hard nail keratin Ha-1.
Expression of this keratin defines the proximal (A) and distal (B) margins of the matrix.
FIGURE 10 Continued.
FIGURE 11A Histological section stained with hematoxylin and eosin (×10). Longitudinal biopsy sample of big toenail. When a toenail (np)
becomes thickened and rigid (onychogryphosis), this may reflect an altered shape of the matrix (m) and proximal nail fold as seen in a normal
section (Fig. 3).
FIGURE 11B Histological section (×10). Longitudinal biopsy sample of big toenail in Fig. 11A with onychogryphosis. The antibody Tric-1 to
the hard keratin Ha-1 illustrates the altered relationship between the matrix and the nail fold associated with onychogryphosis.
399
FIGURE 12 Longitudinal biopsy sample of nail with onychogryphosis (Fig. 11 A), labeled with the monoclonal antibody Tric-1 (Fig. 11B).
Distal matrix keratinocytes have altered organization, losing a longitudinal gradient of hard keratin expression.
In the dermis, vimentin was strongly positive in fibroblasts and vascular

endothelial cells. Vimentin and desmin were expressed in the smooth muscle
wall of some vessels. The S100 stain, for cells of neural crest origin, revealed
perivascular nerves, glomus bodies, and Meissner’s corpuscles distally.
Filaggrin could not be demonstrated in the matrix in Haneke’s work or by

electron microscopy [10]. However, Manabe and O’Guin [44] have detected the
coexistence of trichohyalin and filaggrin in monkey nail, located in the area they
term the “dorsal matrix,” which is likely to correspond to the most proximal
aspect of the human nail matrix as it merges with the undersurface of the
proximal nail fold. Kitahara and Ogawa [31] have identified filaggrin in the
human nail in the same location, and O’Keefe et al. [45] have found trichohyalin
in the “ventral matrix” of human nail, which is synonymous with the nail bed.
Manabe noted that these two proteins coexist with keratins 6 and 16, which are
more characteristic of nail bed than of matrix. It is argued that filaggrin and
trichohyalin may act to stabilize the intermediate filament network of keratins 6
and 16, which are normally associated with unstable or hyperproliferative states.
The plasminogen activator inhibitor PAI- 2 has been detected in the nail bed
and matrix, where it has been argued that it may have a role in protecting against
programmed cell death [46].
The basement membrane zone of the entire nail unit has been examined by
means of a wide range of monoclonal and polyclonal antibodies [25]. Collagen
VII, fibronectin, chondroitin sulfate, and tenascin were among the antigens
detected. All except tenascin were present in a quantity and pattern
indistinguishable from that characteristic of normal skin. Tenascin was absent
from the nail bed, which was attributed to the fact that the dermal papillae are
altered or considered absent.
1.5
Organization of Keratin Fibrils in Nail Cells
The keratin fibrils in hairs are oriented along the hair axis. In trend setting X-ray
diffraction studies of proteins from different biological sources, Astbury and
Sissons [47], as well as in the later studies of Derksen et al, [48], it was
demonstrated that the main bulk of keratin fibrils is oriented perpendicular to the
growth axis of the nail plate (i.e., mainly in the y-axis direction). More recently,
this finding was confirmed by Baden [49] and in more detail by Forslind [50]
(cf. Sec. 2.2 X-ray studies).
1.6
Nail Growth
Nail growth derives from nail matrix and can be assessed at a tissue level or
macroscopically in terms of nail length, mass, or thickness.
1.6.1
Markers of Matrix and Nail Bed Proliferation
Lewis [3] proposed that there was a significant contribution to nail plate
production from the nail bed and the ventral aspect of the proximal nail
fold. Zaias and Alvarez [7] disagreed with Lewis on the basis of in vivo
autoradiographic work on squirrel monkeys in which dynamic aspects of the
process were examined. Tritiated thymidine injected into experimental animals
was incorporated only into classical matrix (or intermediate matrix, to use
Lewis’s terminology). Norton used human subjects in further autoradiographic
studies [17]. Although there was some incorporation of radiolabeled glycine in
the area of the nail bed, it was in a poorly defined location, making clear
statements impossible.
Antibodies to proliferating cell nuclear antigen and to the antigen KI-67
associated with cell cycling, have been used on longitudinal sections of healthy
and diseased nail units (Fig. 6) [16]. Both markers demonstrated labeling indices
in excess of 20% for the nail matrix, in contrast with 1 % or less for the nail bed
in healthy tissue. In psoriatic nail and onychomycosis, the labeling index of nail
bed rises above 29%. While these indices do not directly measure nail plate
production, a very low index for normal nail bed is consistent with results of
other studies suggesting that the nail bed is insignificant in normal nail
production. The situation may change in disease, and definition of nail plate
becomes difficult when substantial subungual hyperkeratosis produces a ventral
nail of indeterminate character [51, 52].
1.6.2
Nail Plate Indicators of Matrix Location
Johnson et al. [53, 54] believed that the nail bed produces a significant fraction
of the nail plate. They examined nail growth by measuring change in nail
thickness along a proximal-to-distal longitudinal axis and demonstrated that 21 %
of nail plate thickness in traumatically lost big toenails was gained as the nail
grew over the nail bed. This was taken as evidence of nail bed contribution to the
nail plate.
A similar study developed this observation with histology of the nail plate
taken at fixed reference points along the longitudinal nail axis and comparing
nail plate thickness at these sites with numbers of corneocytes in the
dorsoventral axis of the nail [16]. The result was to confirm the observation that
the nail plate thickens over the nail bed but that this thickening is not matched by
an increase in nail cells. In fact, the number of cells reduces by 10%, but this was
not of statistical significance. These combined studies may be reconciled if we
propose that the shape of cells within the big toenail becomes altered with
compaction as the nail grows. This is a likely explanation for the development of
transverse rippling in nails in the presence of habitual distal trauma, as noted in
clinical experience.
1.6.3
Ultrasonography as a Tool to Define Nail Matrix
Ultrasound studies of the nail plate have done little to support the notion that the
nail bed contributes significantly to its substance [55, 56]. Jemec and Serup [56]
claimed that the nail plate had a clear two-part structure, none of which appeared
to come from the nail bed. Finlay et al. [55] observed that the nail plate had a more
rapid ultrasound transmission distally, a paradoxical finding if one imagines a
nail bed contribution. This last comment is almost diametrically opposite that of
Johnson et al. [54].
1.6.4
Macroscopic Nail Growth
Nail growth is slow compared with scalp hair growth. Observations of nail
growth have not attained the same interest as malformation and brittleness,
although it may be characteristic of some conditions such as yellow nail syndrome.
Most studies on nail growth have been, in this context, short-term studies on
variable numbers of individuals differing in age and sex [57, 58]. For the
description of an outstanding and truly longitudinal study of nail growth in one
and the same individual, the reader is referred to the report of William Bean
[59], which provides a lucid review on the subject.
The remarkable constancy recorded in fingernail growth is emphasized on
reviewing the literature. Not considering the variations due to age [59], the
average normal growth rate is 0.1 mm/day, still which is comparable to the
growth of vellus hairs on the loins of women. Except when a person is subject to
malnutrition and/or serious disease, the growth rate remains approximately
constant. Nail biters, however, are reported to have a growth rate speeded up by
10 to 20% [58, 60]. The same amount of increase was noted in psoriasis [61], a
skin disease associated with an increased epidermal turnover also in clinically
unaffected skin. Few things seem to influence the normal growth rate of nails
except fever and different kinds of malnutrition. There are slight variations in the
speed of nail growth of different fingers, but these are not consistently
significant.
Most authors report that there is no significant influence on growth in
temperate zones of the world by climate, season, or geographical location,
FIGURE 13 Nail growth can be measured by making a transverse groove in the dorsal nail
surface, abutting the lunula. Black ink in the groove improves the photographic record. A
second groove is made to measure the distance grown over a suitable interval. (e.g., 1
month).
although the literature contains some data in conflict with this opinion [59]. The
speed of nail growth diminishes with advancing age [59]. In contrast, data on
toenails seem to be more at variance [60].
1.6.5
How to Measure Nail Growth
Measurements of nail growth are easily performed. The distal part of the nail
crescent, the lunula (Fig. 13), represents the distal part of the nail matrix, which
is stationary. The lunula generally is pale and whitish in Caucasians and
Mongolians, whereas the more distal nail bed gives a characteristic pink shade to
the nail plate. In other words, there is a clearly visible demarcation line between
the lunula and the nail bed that does not change its anatomical position. A notch
is cut into the nail plate at this line, and after a suitable time, 6 to 12 weeks later,
the distal movement of the nail plate can be assessed by measuring the distance
from the notch to the lunula. The actual measurement is made easy by making a
new notch at the lunula and impregnating both notches with Indian ink.
(Fig. 13). Measurement is facilitated by using a dermatoscope with integral
scale. With a digital camera and appropriate software as well, record keeping and
measurement can be combined. The problem with this technique is that the
lunula is not always visible in all digits. Almost universally seen in the thumb, it
becomes progressively more concealed beneath the proximal nail fold in the
more lateral digits. It is rarely seen in the toes. To measure growth in digits with
no apparent lunula, the proximal nail fold must to be used as a substitute, and
this is problematic when the structure has experienced inflammation or trauma,
which can alter its dimensions.
1.6.6
Movement of the Nail Bed
A common observation is that subungual hemorrhages move along with the
growing nail plate. In an experimental half-nail study, Zaias [63] has shown that
the nail bed does not actually move distally by itself. The distal movement of
subungual hemorrhage is thus due to its adherence to the moving nail plate.
2
EXPERIMENTAL BIOPHYSICAL STUDIES ON
FINGER NAILS
2.1
Considerations on the Structure-Function Relationships at
Different Structural Levels of the Nail
The studies of human fingernails in our laboratory have aimed at interpreting the
physical properties of fingernails related to the architecture at the cellular and
macromolecular levels. As a starting point, we decided that we should gain
thorough knowledge of the normal nail histology and the structural organization
of the cell contacts as well as the organization of the intracellular fibrous
component. This was considered to be essential for an explanation of the
functional properties of nails in relation to their structure at different levels of
resolution. It can be foreseen that such information will provide a basis for a
better understanding of pathological conditions in nails.
2.2
X-Ray Diffraction Studies of Normal Human Fingernails
Using the light microscope, routine controls of nail clippings with oblique
incident and reflected light revealed that the nail plate can be separated into two
entities. The dorsal nail plate appeared to be homogeneous in the cross section,
whereas the ventral nail plate showed oblique compression lines that actually
changed form during observation in a preparation microscope immediately after
the cutting procedure. Thus the changes observed represented artifacts imposed
by the shear forces of the scissors’ edges that compressed the plastic material of
the ventral nail plate, causing the appearance of an oblique lamellar structure.
This observation suggested that the architecture of the nail plate may indeed be
different in the two parts of the nail.
Laminar features of nail plate construction can also be seen at the free edge,
where there is a combination of cutting and chronic low grade trauma (Fig. 14).
At a histological level, vertical sections of nail plate show compacted
onychocytes in all layers, with greater compaction dorsally (Fig. 15). Some of
these ghost cells retain nuclear remnants reflecting the incomplete condensation
of the nucleus with matrix maturation, in constrast to normal skin, where

maturation proceeds through the level of a granular layer. These remanants can
also be seen in horizontal preparations through the dorsum of the nail (Fig. 15).
2.2.1
The Experimental Apparatus
To study the fiber orientation in nails, a Chesley micro X-ray diffraction camera
was used. It was collimated with a lead glass capillary with a diameter of 300 µm,
which defined the cross section of the X-ray beam that explored the irradiated
nail volume . The Chesley camera has a specimento-film distance of 15 mm,
allowing a resolution of repeating structural units in the specimen with spacings
shorter than 1.5 nm when CuK radiation, having a wavelength of 1.54 Å (0.154
nm), is used. In a sequence of experiments, the nail clippings were oriented so
that all possible planes were represented in different diffractograms. The Chesley
camera allowed diffractograms to be obtained from the dorsal and ventral nail
plate separately. Both specimens were subjected to X-ray diffraction before and
after a dissection separating the dorsal from the ventral nail plate. To
record possible short spacings representing long repeating distances in the
specimen, some specimens were also exposed in a flat film camera with the same
collimation but with a specimen-to-film distance of 50 mm, thus allowing for a
higher resolution. This made it possible to identify all maxima with high
precision.
2.2.2
The Material
The material of this study consisted of nail clippings from persons ranging in age
from 2 to 50 years. Nails from two Rhesus monkeys (Macaca mulatta) were also
used.
2.2.3
Interpretation of X-Ray Data
The X-ray diffraction pattern showed different degrees of orientation depending
on the direction of the incident X-ray beam in relation to the growth (x) axis, as
had been observed earlier [48]. With the incident beam coinciding with the x
axis, a pattern showing good order was obtained from the nail clippings,
indicating a main fiber orientation along the y axis. With the X-ray beam
incident along the y and z axis, respectively, a lesser degree of order was
revealed. No significant differences between nails from children or from adults
were seen, and this was true also when human and monkey nails were compared
[50].
FIGURE 14 Longitudinal section of distal nail plate illustrating the free edge at two magnifications: (A) × 75 and (B) × 150. The distal dorsal
surface becomes fragmented and splits, revealing the underlying laminar nail structure.
407
FIGURE 15 Dorsal view of nail plate, mounted in diphenylxanthine and viewed with crossed polarized filters at two magnifications: (A) ×200
and (B) ×400. Nuclear remnants are visible, characteristic of the sequence of matrix differentiation omitting a granular layer.
409
A refinement of the old diffraction studies was possible by the fine collimation
of the Chesley camera, which allowed separate recordings from the dorsal and
the ventral nail plates without dissection of the, specimens. As a control,
horizontal separation of the ventral nail plate from the dorsal (in the x/y plane)
was done with a razor blade, which proved that the camera collimation actually
allows differential analysis of the two nail plate units.
The degree of orientation in the X-ray diffraction pattern was shown to be
related to the different portions of the nail plate. Patterns from the different
portions of the nail plate and the isolated ventral nail plate had the same general
features as the patterns from compound nail clippings. However, with the
incident X-ray beam parallel to the x axis, the ventral nail plate revealed a very
high degree of fiber orientation. In fact, this pattern much resembled that of a
fiber diffraction pattern obtained from a single hair fibre.
Although a considerable degree of orientation was detected in the dorsal nail
plate, a dispersion of the fibrils was at hand, as revealed by the more pronounced
arcing of the diffraction maxima. These findings were observed both in the
dissected nail plates and in selected-area diffraction experiments performed on
compound nail clippings.
In the conspicuously well-oriented X-ray diffraction pattern from the ventral
nail plate, the typical -keratin pattern of meridional reflections at
TABLE 1 Important X-Ray Diffraction Repeat Distances (Å) Recorded in Nails

Naila Intensity Porcupine quill tip
Observed equatorial 27 Strong 27b
reflections
8.8–10.8 Strong 9.2–10.5
Observed 23 Medium-weak 24.5c
meridional
reflections
5.2 Strong 5.18
4.3 Medium-strong 4.2
3.9 Medium 3.9d
aData from Ref. 50.
bData from G.Swanbeck. In: GN Ramachandran, ed. Aspects of Protein Structure. New
York: Academic Press, 1963, pp 93–101.
cData from A Liang. Acta Crystallogr 9:446–451, 1965.
dData from WT Astbury, JW Hagg. Th. Biochim Biophys Acta 10:483–490, 1953.
3.9, 4.3, and 5.2 Å was clearly seen. At the equator (the horizontal axis at right
angle to the fiber axis) the corresponding -keratin reflections representing
spacings of 8.8–10.8 Å and 27 Å spacings were discerned (Table 1).
2.3
Transmission Electron Microscopic Investigation
The artifacts imposed by a pair of scissors on the ventral nail plate and the results
of the diffraction study called for further studies on the architecture of the nail
plate. Therefore human nail clippings and small dissected tissue blocks from
monkey nail roots were fixed in osmium tetroxide and prepared for electron
microscopy according to standard methods [50]. The sections were taken
perpendicular to the nail plate surface and along the growth axis (the x/z plane).
At low magnification the information gained confirmed the results from
earlier light microscope investigations. In the electron micrographs at a primary
magnification below 4000 times, little structural detail was seen except for the cell
membranes. This is due to the very dense and homogeneous keratin material of
the mature nail. The cells of the dorsal nail plate appeared very flat, with their
smallest diameter perpendicular to the nail surface (in the z-axis direction). The
cells of the dorsal nail plate were closely connected by an electron-dense
substance that filled the intercellular spaces. Sometimes the central dense line
between the cells was bisected by a faint intermediate line. The total width of
this junction measured up to 25 nm.
In contrast to the almost straight cell border of the dorsal nail plate, the cells of
the ventral nail plate exposed a much more meandering outline in the section.
The height of the cells in the ventral nail plate was several times that of the cells
in the dorsal nail plate. Where these cells were joined by a dense intercellular
substance, the space between the cells was approximately 20 nm.
At high magnification a preferred orientation of the keratin filament in the y-
axis direction could be recognized in the ventral nail plate, whereas the fibrils of
the dorsal nail plate were seen to be running in several directions.
In the electron micrographs the histochemically demonstrated border between
the dorsal and the ventral nail plates [64], which is also seen at the cut surface of
the nail clippings (Fig. 16), was not obvious. Rather, the differentiation
characteristics of these two layers were achieved by an almost continuous change
in the cell adhesion pattern. Juvenile and adult human nails as well as monkey nails
were similar with respect to the organization of cells and the subcellular
components as seen in electron micrographs.
FIGURE 16 Vertical section through a longitudinal sample of nail plate. Viewed by means of crossed polarized filters to illustrate the laminar
organization of the structure and the different characteristics in dorsal and ventral nail.
413
2.4
Structure-Function Relationships in Nails: Interpretation
of X-Ray Diffraction and Electron Microscopic Data
The mechanical properties of the compound nail plate can be explained on the
basis of the architecture at the cellular and macromolecular levels by the
following arguments. In the dorsal nail plate it is conceivable that the
intercellular junctions permit good interaction between cells, thus permitting a
highly rigid plate to be formed. In contrast, the interlocking cell borders of the
ventral nail plate denote a large cell surface in comparison with cell volume. The
attachments of cells appear at discrete points on the cell surface, leaving open
spaces between cells. Consequently this part of the nail plate shows more plastic
properties than are observed in the dorsal nail plate.
Referring to the dorsal nail plate, it is interesting to note that the intercellular
coupling of the cortex cells in hair fibers appears to be very similar to that of the
cell coupling in the dorsal nail plate in terms of widths and continuity, as
revealed by the electron micrographs. In the hair, the cell coupling provides for
good interaction between cortex cells, which makes fiber recover after bending
or tensional stress more or less complete.
The X-ray diffraction patterns clearly show that the nail contains the fibrous -
keratin. In the ventral nail plate there is a very high degree of order (i.e., a
preferred orientation of the keratin fibrils), with the X-ray beam coinciding with
the x axis. When the X-ray beam coincides with any of the other two axes of our
coordinate system, the spread in angular distribution of maxima in the
diffractogram (i.e., wider arcs) suggests a lesser degree of the orientation of the
keratin fibrils. These findings support the concept of a fibril arrangement that
will provide for the torsional rigidity of the nail plate and high breaking strength.
Expressed in another way, the greater angular dispersion of the keratin fibril
orientation means that the fibrils will be able to take up forces imposed on the
keratinized cells from different directions; that is, the fibril function is to provide
an internal reinforcement for the cells. The very pronounced fibril orientation
perpendicular to the growth (x) axis restricted to the plane of the nail surface
(i.e., in the y-axis direction) will provide a reinforcement that can prevent
cleaving of the nail in a specific direction (along the x axis) toward the root. If such
cleaving occurs, it may cause permanent damage to the root and result in a
permanently deficient nail.
Our investigations indicate functional relationships between the cellular
arrangement, the cell junction types, and the structure of the nail plate. At the
macroscopic level, the cutting property of a fingernail can be ascribed to the
arrangement of the dorsal and the ventral nail plates. It can easily be observed
that the dorsal nail plate per se is rather brittle and that the nail plate as a whole
will gain in strength by the cooperation of the dorsal and the ventral nail plates.
The method employed for making sharp razor blades by enclosure of a very hard
and brittle core between two sheets of a comparatively plastic material will
illustrate the function of composite design. In the razor blade, the material
forming the central core and the cutting edge is very thin and brittle. The necessary
rigidity of the razor blade is attained by enclosure in a non brittle, easily
deformable material that can take strain under stress relaxation due to plastic
flow. This allows bending deformation without undue stresses in the brittle
material of high elastic modulus.
In the composite nail, the brittle material of the razor blade corresponds to the
dorsal nail plate, whereas the nonbrittle, easily deformable material corresponds
to the ventral nail plate, which has plastic properties. In the nail plate the most
important force vector has a palmar-dorsal direction, (i.e., coinciding with the z
axis), as can be realized when nails are used for scratching. Consequently, the
deformable material is positioned on the palmar aspect of the brittle dorsal nail
plate. Impact of forces in a dorsal-palmar direction will be distributed onto the
supporting subungular tissues, and in nails of normal length bending will be
relatively unimportant.
The transverse curvature in the y and z planes and the longitudinal curvature in
the x and z planes of a normal nail will give a platelike structure to the nail,
impacting a considerable gain in load-carrying capacity and preventing buckling.
It may be easily observed that persons with slender fingers and marked
longitudinal and transversal curvatures have less tendency to break their nails
than persons with flat, barely curved nail plates.
The longitudinal curvature (in the x and z planes) in the normal nail plate
could be due to difference in growth rates of the cells of the nail plate and/or to
pressure forming the curvature. Such a pressure-induced curvature would in the
present case originate from the pressure of the tissue proximal to the eponychium,
which constitutes the roof of the proximal nail fold and will oppose the upward
pressure exerted by the dividing cells of the growing nail. This connective tissue
forms what could be called a “dorsal band”of oriented collagen fibrils that have
ventral connections with the periostium of the distant phalanx and collectively
serve as an anchorage for the connected tissue. As was pointed out earlier, the
cells leaving the root from the bottom of the nail fold will thus be exposed to the
constant forces of the dividing proximal cells counteracted by the pressure of the
connective tissue “dorsal band.” It is likely that such forces will introduce an
orientation of the growing cells, leading to the lamellar shape of the cell
aggregates as seen in vertical sections (in the z/x plane). In the nail root, the more
distal the origin of the cells forming the hard nail plate, the less will be the
pressure of the “dorsal band” that opposes the pressure of growth, and
consequently the lower the tendency of the cells to assume a finite lamellar form.
As seen in the electron microscope, the vertical section through the nail plate
fully agrees with this interpretation [50]. Such a pattern of growth will produce
an apparent difference in growth speed of the cells because the dorsal nail plate
cells will be flattened to a higher degree than those of the lower part of the
ventral nail plate. Ample support of this interpretation is given by the

autoradiographic experiments of Zaias and Alvarez [7], as well as in a report by
Maibach and Epstein [65].
2.5
Nail Constituents and Their Significance
2.5.1
Comments on the “Hardness” of Nails: Sulfur and Calcium
Content
In literature as well as in everyday life, the term “hardness” is used to describe a
physical property of fingernails that should be rightly defined as rigidity or
stiffness. Later in this chapter we will also discuss how this physical property of
rigidity may be measured and what factors influence it. Since nails contain hard
keratins, a high content of sulfur is to be expected in this tissue.The cystine of
mature nails is roughly 9% of the weight [66–68], which is a relatively high
value compared with 1 % in callus and 4.1 wt% in stratum corneum disjunctum
(Table 2.). Measured as the sulfur content related to the dry weight, this value
corresponds roughly to 3.82 wt% sulfur. Low cystine (and consequently low
sulfur) values are found in diseases of genetic origin such as trichohiodystrophy
[69].
TABLE 2 Content of Some Important Substances in Skin and Its Appendages Expressed
as Percentage of Weight
Stratum corneum Hair Nail
Water 6.5 8.6 10.5
Calcium 0.04 0.02–0.5 0.1–0.2a
Cystineb 3.8 15.5 12.0
4.1c 15.1c 9.4c
aAtomic absorption analysis data [50].
bCystine determined as cysteine.
cData from Ref. 67.
Source: Data compiled from S.Rothman. Physiology and Biochemistry of the Skin.
Chicago: University of Chicago Press, 1954.
The cystine and sulfur contents of nails depend on a normal nutritional intake
and status. Thus iron deficiency may result in a diminished cystine content [66, 70].
Attempts to obtain data on the “hardness” of nails have been made by
indentation hardness techniques and by other physical means [49, 71–73]. In
view of the data we have presented [74], reviewed shortly, such investigations
have been inconclusive.
The “hardness” of the nail has also been attributed to an alleged high content
of calcium. No quantitative data to support this proposal have been published
[50, 75].
2.5.2
The Water Content of Nails
It is an everyday experience that nails become more pliable when soaked in
water. When completely dry, the nail becomes brittle and has weak mechanical
properties. The water loss through the nail plate has been measured to be 2.40mg/
cm2/hr [76], whereas the water loss through unaffected skin was 0.8 mg/cm2/hr,
as recalculated from Nilsson [77]. This means that the water loss through the nail
plate is comparable to that of eczematous skin (i.e., > 5 mg/cm2/hr [49]. Thus the
nail plate represents a very poor barrier to water loss. We may draw two
conclusions:
1. A hydrated nail plate has the required elasticity and flexibility to function as
an indispensable tool.
2. Leakage of water through the nail plate ensures that it will have these
desired mechanical properties.
2.5.3
Dry Mass and Sulfur Determination
Our proposed explanation for the rigidity of the nail plate does not consider any
possible differences in dry mass or elemental composition of the cells in the two
morphological entities. To reveal whether any possible differences in dry mass
and/or sulfur content could contribute to the nail plate rigidity, a
microradiographic study of normal human nail was undertaken. Quantitative
microradiography allows both dry mass determination and sulfur determination
on previously untreated freeze-sectioned and freeze-dried specimens.
In this method nail sections with an approximate thickness of 20 µm were
mounted parallel to a reference system in close contact with a finegrained, X-ray-
sensitive emulsion. The reference system has an elemental composition
approximating organic material and consists of a step-wedge of Mylar film. The
mounting allows the specimen and the reference system to be simultaneously
exposed to continuous X-rays having a wavelength of 5 to 20 Å for quantitative
dry weight determination [78]. Microdensitometry of the X-ray absorption
images produced by the specimen and the stepwedge, respectively, provided data
for the dry mass determination.
For a subsequent sulfur determination, the same specimen exposed to strictly
monochromatic X-rays produced two micrographs recorded at two different
wave lengths (Ru-L , 4.846 Å; Mo-L , 5.177 Å), one on each side of the K-
absorption edge of sulfur (5.018 Å). Microdensitometry allows a determination of
the absorption differences registered in the two micrographs, and this difference
reflects the relative content of sulfur per unit area. Combining these data with
those of the dry weight determination allows the calculation of the percentage by
weight of sulfur [79].
After statistical analysis of the microradiographic dry mass and the sulfur
data, it could be stated that no conspicuous dry mass differences were recorded
when the dorsal and the ventral nail plates were compared. The correspondence
of the mass values of the dorsal nail plate and the ventral nail plate thus supports
the proposed explanation of a microarchitectural organization of fibrous keratin
and cell organization to explain the nail “hardness”. The increase in dry mass of
about fourfold from a level adjacent to the matrix cells up to the mature dorsal
nail plate is also consistent with the electron microscopic picture, which shows
the mature cell to be completely filled with fibrous material.
The preliminary X-ray microradiographic sulfur analysis suggests a close
relation between the dry mass of keratin and the relative sulfur content, which is
between 3 and 6 wt% [79]. This corresponds to the amino acid analysis, which
has given a cystine content of 9.4% corresponding approximately to 2.4% of
sulfur. The lower value obtained in the amino acid analysis may be due to losses
of material in the preparation procedures preceding the amino acid analysis.
Since, however, the X-ray microradiographic sulfur data were, based on a
comparatively small number of measurements, a conclusive statement of the
absolute sulfur content in human nails cannot be given with this method. In a
subsequent X-ray microanalytical study, the sulfur content of nails was shown to
be 3.8 wt% [80],
2.5.4
Scanning Electron Microscope Studies: Support for the
Proposed Explanation of Nail Rigidity
As stated earlier, the suggested explanation of the rigidity of finger nails implies
that the dorsal nail plate is comparatively brittle, whereas the supporting ventral
nail plate has plastic properties at deformation. A simple experiment would be to
cut a nail in the x/z planes from the dorsal and the ventral aspect of the nail,
respectively. In the first case the strain will be dissipated through the plastic
material of the supporting ventral nail plate and a smooth surface of the cut will
result.
In the second case, where the cut is running from the ventral nail plate toward
the dorsal, the plastic material of the ventral nail plate is expected to deform
smoothly under the shear forces of the sharp edge. The surface of the ventral nail
plate will thus be cut with a comparatively even surface with no conspicuous
cracks. The dorsal nail plate, which is to be regarded as a material of
comparatively high modulus of elasticity, will give rise to a considerable
accumulation of elastic strain in front of the edge before the rupture in the
material occurs. The material in front of the scalpel’s edge will thus separate,
discontinuously producing cracks of different widths depending on the natural

notches in the anisotropic material (i.e., the lamellar cellular structure). When a
scanning electron microscope (SEM) was used to study the details of the cut
surfaces, these assumptions were validated. The difference observed between
two cutting directions from the plastic toward the elastic material and vice versa
confirms the contention as stated in the preceding paragraph [81].
2.5.5
Analysis of Calcium by Atomic Absorption Spectroscopy
The hardness of fingernails has in the past been attributed to an alleged high
content of calcium. To investigate this possible influence of calcium on the
rigidity of human fingernails, nail clippings were collected from all fingers of
seven persons of both sexes, aged 11 to 43 years, none of whom had any known
general or skin diseases. To reduce possible short-term variations, the collection
period covered 8 months. The specimens were minced, and calcium was
extracted either by concentrated hydrochloric acid for 7 days or by dissolution of
clippings in concentrated nitric acid. At atomic absorption spectroscopy the
reference solvent was made identical with that used for calcium extraction. Our
data showed that the calcium content of normal nails was below 0.2 wt % and
could thus be regarded as a trace that does not directly influence the rigidity of
nails. These data were independently confirmed by Vellar [75], who used the
same quantitative method but with different extraction techniques.
2.5.6
X-Ray Microanalysis of Sulfur, Calcium, and Other
Electrolytes in the Nail Plate
Analytical electron microscopy is based on the fact that X-rays are generated in a
specimen during electron microscopy. When bombarded with fast electrons,
atoms in the specimen will lose orbital electrons of the inner shell. The vacancies
are immediately filled with fast electrons from outer shells. The energy
difference between outer and inner shells is released as electromagnetic radiation
(i.e., characteristic X-rays). Thus, a radiation spectrum emanating from all the
elements in the bombarded specimen is obtained. The use of an energy-
dispersive detector to simultaneously record all energies emitted allows
qualitative as well as quantitative analysis of elements in the specimen; this
technique allows a point-to-point resolution of about 1 µm in the localization of
elements.
With the arrival of X-ray microanalysis, a further investigation was undertaken
to determine whether the calcium content of nails is intrinsic or due to absorbed
contaminants. At the same time the technique allowed determination of possible
sulfur gradients in the cross section of the nail, a matter of considerable interest
as seen from the preceding paragraphs. In this new investigation, nail clippings
and freeze-dried nail root sections were used. The latter specimens were obtained
from the distal phalanx of the second toe collected from four corpses of both
sexes (20–40 years). The nail root was dissected free from the bone and
subsequently given support by carboxymethylcellulose gel, instantly frozen in
isopentane cooled by liquid nitrogen, and transferred to a cryostat for freeze-
sectioning at 25°C. The sections obtained were mounted onto carbon plates by
means of a graphite solution. Table 3 gives the measured relative amounts of
sulfur, potassium, and chlorine for the dorsal and the ventral nail plates. There
was a conspicuous difference in the potassium content of the dorsal and the
ventral nail plates, but variation in the potassium content (by a factor of 10) had
been recorded by Harrison and Clementa in a spark source mass spectrometry
study [82]. Analysis of the calcium content in different portions of the human
nail (Table 4) indicated that there are great differences between the dorsal and
ventral surfaces of the nail plate. It is also noticeable that internal, nonexposed
parts of the nail reveal a very low calcium content. Such nonexposed parts
consist of the surface areas at a vertical
TABLE 3 Relative Amounts of Sulfur, Potassium, and Chlorine in Nails Obtained by X-

Ray Microanalysis in the Electron Microscopea
Dorsal nail plate Ventral nail plate
Sulfur 3.84±0.29 (11) 3.93±0.51 (12)
Potassium 0.66±0.14(8) 0.29±0.11 (7)
Chlorine 0.70±0.08 (8) 0.63±0.07 (9)
aMean vaues ± standard error. Numbers in parentheses indicate numbers of specimens.
Pooled data from nail clippings and frozen sections.

Source: Ref. 80.
cut in the z/x plane that have not been in direct contact with the environment. In
view of the data obtainable from literature and from our past experience we
suggested [80] that the differences recorded were due to a steep gradient at the
surface of a nail plate, where the calcium content could vary within an order of
magnitude. In the bulk of the nail material, on the other hand, the calcium content
is approximately constant. As a result of the cellular architecture of the two
surfaces (i.e., the dorsal and the ventral nail plate), the nail offers different areas
for adsorption at these respective surfaces. The physical-chemical basis for such
adsorption is the readiness of calcium ions to exchange for protons. We thus
propose that the nail surface matrix may act as a kind of ion exchanger, a
property consistent with the observed data and with previously recorded
considerable seasonal and individual variations in the calcium content of nails.
2.5.7
The Determination of the Rigidity of Human Fingernails
In a further attempt to test the validity of the hypothesis that the rigidity of the
human fingernail plate was related to a composite structure of a dorsal
TABLE 4 Relative Amounts of Calciuma inDifferent Portions of Human Nails Obtained

by X-Ray Microanalysis in the Electron Microscope
Dorsal surface of nail exposed to environment 1.07±0.19 (16)
Ventral surface of nail exposed to environment 1.90±0.46 (6)
Nonexposed (internal) parts, made available by sectioning nail 0.24±0.06 (7)
aMean vaues ± standard error. Numbers in parentheses indicate number of specimens.
Source: Ref. 80.
nail plate of high elastic modulus and a ventral nail plate with plastic
deformation properties, a series of load experiments was carried out.
Nail clippings were trimmed down to rectangular specimens of 2×5 mm2 and
placed on stainless steel supports at each end. The load was then attached to the
midsection of the specimen. The bending of the nail was measured as a function
of the load imposed on the section. The bending deformation was measured by a
metallurgist’s microscope mounted on a fine division threading, which allowed a
precision in the measurement of 0.05 µm.
From the load-bending diagrams “the effective elastic modulus” of the
specimens could be calculated. This measured property corresponds to a
numerical evaluation of a property that describes the rigidity (or stiffness) of the
nail plate.
The measurements were made on specimens carefully equilibrated to different
relative humidities over periods of more than 14 days and on specimens that had
been dried for an hour at 90°C and subsequently at 60°C for 14 days. Neither the
thickness nor the width of the specimens had any significant influence on the
effective elastic modulus. The same holds true for the direction of the natural
nail curvature (i.e., whether the specimen was applied in the loading device with
the convex side up or down). The effective elastic modulus was strongly
dependent, however, on the water content of the nail specimen or on
pretreatment with organic solvents, detergents, mineral oils, and so on. These
findings are in harmony with earlier reports [71]. Constant loading of the nail
revealed a nonlinear deflection during the first 2 hr, after which the deflection
rate was approximately linear. Applying successively increasing loads to the
specimen by successive addition of weights resulted in a linear relationship of
load to deflection. When the final accumulated load was removed in one step, the
deflection curve decreased nonlinearly. The main part of the recovery occurred
within a small fraction of time,- a complete recovery required more than 17 hr.
Several phenomena, among which at least one elastic and one viscous phase can
be identified, explain this complex behavior of the nail under load.
The (hysteresis) form of the recovery curve with respect to time suggests that
the unloading viscosity is greater than the loading one. The constant load
experiment suggests that several molecular mechanisms are involved in the
deformation process. Among possible effects, one of importance is the movement
of keratin filaments in a nonstructured matrix of the intracellular compartment,
which is less restricted when water is present in the matrix.
The “cementing” material of the intercellular substance will probably also
contribute to the viscosity of the material. These proteins and/or
mucopolysaccharide proteins are likely to be in a random coil state. During
loading, the material of the nail will be oriented in the direction of the
applied stress, and consequently these random coils will be stretched. During the
recovery process after unloading, these polymers strive to regain their random
configuration. This is a time-dependent process that depends, also, on the
viscosity the polymers experience during recovery. In other words, a certain
degree of memory will be retained in the stretched (oriented) molecules. The
return to random coil may be recovered as an increased degree of viscosity.
Thus the effective elastic modulus is a well-defined mechanical property in
this context because the linear range of the load-deformation relationship is
finite. It corresponds to a property that is best described by words such as
“rigidity” or “stiffness,” whereas “hardness” should not be used for a material
that exhibits viscous flow at loading.
Moreover, if the elastic modulus of the nail plate is to be measured in situ by
ultrasound methods, the water content must be determined precisely. With the data
available at present, it is hardly possible to evaluate the influence on the
measured results due to a water gradient in the intact nail plate in situ.
Previous contacts with detergents, organic solvents, oils, and so on may affect
the nail stiffness. This implies that considerable efforts must be made to get a
complete case history from the patient when rigidity (or stiffness) of nails is to be
measured [74].
2.6
Organic and Metabolic Nail Constituents
Endogenous and exogenous materials may be incorporated into keratinocytes.
Some of these exogenous materials will be absorbed directly and some will be
integrated after ingestion or as metabolites. Hair keratinocytes will provide a
time-correlated record of uptake that may depend on the individual capacity for
absorption and environmental and metabolic variations in the availability of the
substance in question. Nails have been used for this kind of analysis to a lesser
extent, probably owing to the slow growth (only a third of that of scalp hairs) and
the obvious great risk for contamination. However, one of the strengths of this
medium is that nail is a less labile source of information than blood—the main
alternative. The glycosylated globin molecule, used for estimation of long-term
diabetic control, has been used as a model in studies measuring nail furosine in
diabetes mellitus. The nail fructose-lysine content is raised in this disease and
has shown a correlation with the severity of diabetic retinopathy and neuropathy
[83]. Nail furosine levels have also shown a good correlation with fasting
glucose and may even compete with glycosylated hemoglobin as an indicator of
long-term diabetic control [84].
Sometimes nail material can be used in screening for relatively uncommon
conditions. Analysis of chloride in nail clippings of a juvenile control population
and in young cystic fibrosis patients revealed a significant increase of chloride,
by a factor of 5, in the latter. This has led to the suggestion of “screening nails by
mail” for inaccessible geographical regions, where sending nails would be
relatively easy.
Steroid sulfatase and its substrate, cholesterol sulfate, were assayed in the nails
of children being screened for X-linked ichthyosis and were found to have
adequate sensitivity and accuracy to be useful [85–87]. Sudan IV-positive
material in nails has been measured as a guide to serum triglycerides [88].
Selenium is a trace element critical for the activity of glutathione peroxidase,
which may protect DNA and other cellular molecules against oxidative damage.
High concentrations are seen to protect against the action of certain carcinogens
in some animal models, and consequently its role in human cancers has been
explored. Analysis of the selenium levels of different rat tissues suggests that
blood selenium may be the best indirect measure of liver selenium, and nail
selenium may best reflect whole-body levels and the level in skeletal and heart
muscle [89]. Nail selenium levels in those being screened for oral cancer [90]
and carcinoma of the breast [85, 91] showed no significant differences between
affected and control patients. However, in a prospective study, toenail selenium
levels had a weak predictive value for the development of advanced prostate
cancer, where low levels of selenium predisposed men to this malignancy [92].
Examination of a wide range of trace elements in the nails of women with breast
cancer failed to show any difference from normal controls [93], and analysis of
nail for zinc showed no significant difference between pellagra patients with low
serum zinc and normal controls [94].
Nail clippings can be used as a source of DNA in forensic work after
amplification by the polymerase chain reaction. Early work required 20 to 30 mg
of nail [95], but this figure has decreased to 9 mg, where the DNA for the HLA-
DQa alleles is used to assess homology with blood samples [96].
2.7
Exogenous Materials in Nail Analysis
Exogenous materials can be considered in two groups: environmental and
ingested substances. In the first category, cadmium, copper, lead, and zinc have
been examined in the hair and nails of young children [97]. This was done to
gauge the exposure to these substances sustained in rural and industrialized areas
of Germany. Both hair and nail reflected the different environments, although the
multiple correlation coefficient was higher for hair than nails.
Water taken from wells in arsenic-rich rock has resulted in arsenic poisoning
on a major scale in West Bengal, India, over the last 10 years. About 50% of
ingested arsenic is excreted in the urine, with smaller amounts in the feces, hair,
and nails. Nail analysis has been used in the Bengal population as well as in
other populations suffering arsenic poisoning. Levels were estimated by using
flow injection hydride generation atomic absorption spectroscopy, which allows
analysis of very small samples and enables comparisons between different
tissues. The Bengal experience suggests that there are similar concentrations in
hair and nail, with a trend toward higher concentrations in the latter [98]. During
an episode of arsenic poisoning in Alaska, the level of arsenic in nail was four
times that found in hair [99]. A study in New Hampshire found that in subjects
drinking from arsenic-rich wells, there was a doubling of toenail arsenic for a 10-
fold rise in water arsenic content [100].
The features of arsenic poisoning were different in Alaska and Bengal, with
far more cutaneous and systemic signs of toxicity in the Bengal population in
spite of similar levels in body tissues. This was attributed to coexistent dietary
deficiencies and ill health in the Bengalis.
In addition to hair and nail, teeth can act as indicators of long-term unwanted
substances and, in particular, heavy metals. One account suggests that hair
reflects an exposure period of 2 to 5 months, nails 12 to 18 months, and teeth a
far longer period, measured in years [101]. These figures are likely to be subject
to the length of the hair, the site of nail sampling (toe vs finger), and the age of
the subject.
Nickel analysis has been performed to establish occupational exposure [102].
The use of forensic nail drug analysis has been reported in Japan, where over 20,
000 people were arrested for the abuse of methamphetamine in 1987 [102–104].
It was found that the drug enters the nail via both matrix and nail bed. Chronic
drug abusers could be distinguished from those with a single recent ingestion by
scraping the undersurface of the nail before analysis. This would remove the nail
bed contribution and the drug it contained in the “one-off ” abuser.
Simultaneous hair and nail analysis has been performed to compare the
capacity of the tissues to reflect chronic abuse of cocaine [105] and
amphetamines [106]. Miller et al. [105] found that concentrations of cocaine and
its derivatives were higher in hair than in nail, whereas Cirimele et al. [106]
found that the concentrations of amphetamines and its metabolites were similar
in both tissues. Analysis of nail clippings from the newborn by gas
chromatography-mass spectroscopy can provide evidence of exposure to cocaine
during embryogenesis. Given the point of nail formation, it is likely that the
levels will reflect exposure after the fourteenth week [107]. Inclusion via the nail
bed of the antifungal agent terbinafine has also been observed [108]. Access of
the drug to the nail plate via the nail bed may be one of the important factors
allowing effective therapy to be delivered in less time than it takes to grow a nail
[109, 110]. In vitro models for the uptake and delivery of terbinafine by nail
plate have been employed to examine aspects of this process [111].
A single large dose of methamphetamine can be detected by mass
fragmentography in saliva up to 2 days later, in hair up to 18 days, and in nail for
the next 45 days [104]. Chloroquine [112] has also been measured in nail
clippings for research purposes up to a year after ingestion.
To obtain reliable data that may be used for clinical (and forensic) analysis,
data on nails constituents should be related to reference data that have been
obtained from analyses of nails accumulated over a long period, preferably over
a year. Parallel to collection of nail material, other tissue and/or blood samples
should be collected for concomitant analysis.
Careful removal of nail bed material should also be performed to avoid undue
influences on the results of the analyses owing to characteristics of the nail bed
that are more like those of stratum corneum than of nail. When such precautions
have been observed and the range of normal trace element content has been
established, nail may provide an available source of tissue material for clinical
analysis in diagnosis and follow-up of a given therapy.
3
CONCLUSIONS
The authors’ aim has been to demonstrate the applicability of biophysical
approaches and methods in research on a special appendix of the integument, the
nail. In relation to the work done in our laboratories over the past decades, a
concept of the structure and functional properties of normal fingernails has evolved
that may be summarized as follows.
The nail is a composite cellular structure that gains its stiffness from the
organization of the intercellular fibrous protein keratin, as well as from the
cellular architecture of the two parts of the nail plate, the dorsal and the ventral
nail plates. The dorsal nail plate has been shown to be stiff and comparatively
brittle, whereas the ventral nail plate is soft and pliable. Together they form a
rigid structure capable of withstanding bending forces. Thus, the “hardness” of
the nail should rather be called stiffness or rigidity, as can be understood from
the structural considerations presented here. Our biophysical and biochemical
data clearly demonstrate that factors such as the calcium content play an
insignificant role for the stiffness of the nail plate. Nail changes such as splitting
of the nail plate and brittleness are cosmetic problems to which there is presently
no universal cure. It is on the other hand conceivable that exposure to detergents
and organic solvents dissolves the intercellular cementing substance, in as much
as nail plates often split horizontally on such exposures. The susceptibility to
splitting appears to be an individual trait that may also vary over decades.
However, further investigations are necessary to reveal the exact mechanism of
the damage. The nail “strengthening” effect of oils, which undoubtedly increases
the stiffness of the nail plate, also remains to be explained and further explored.
Some of the biological properties and morphology of nails are explained by

the characteristics of the periungual soft tissues. These represent the dynamic and
responsive element of the nail unit that determines the features of nail. While we
explore the physical characteristics of nail, we may explain much by
understanding the biology of the periunguium.
It is obvious from this presentation that nail research is still an undeveloped
part of the research on the human integument and its appendices. It is the authors’
hope that the present text will stimulate to further explorations.
ACKNOWLEDGMENTS
This work is gratefully dedicated to Bo Forslind’s late father, Professor Erik
Forslind, who to a great extent was responsible for Bo’s interest in structure-
function relationships of biological tissues.
Bo Forslind’s experimental work reviewed here was made possible through
the cooperation of a number of enthusiastic colleagues, among whom it is a
special pleasure to mention Professo Björn Afzelius and Godfried Roomans.
Professor Bo Lindström was often of great help to Bo. Excellent technical
assistance was given by Margaretha Andersson and Lennart Wallerman, the
latter having constructed and machined many of the experimental setups. Mrs.
Jenny Bernström provided valuable secretarial help. The interest and
constructive criticism of Bo’s work given over the past decades by many
dermatological colleagues all over the world is gratefully acknowledged.
REFERENCES
1. Le Gros ClarkWE, Buxton Dudley LH. Studies in nail growth. Br J Dermatol
Syphil 1938; 50:221–235.
2. Le Gros Clark WE In: The Tissues of the Body, Clarendon Press, Oxford 1958; 50:
p.308.
3. Lewis BL. Microscopic studies of fetal and mature nail and surounding soft tissue.
Arc Dermatol Syph 1954; 70:732–744.
4. Moll I, Heid HW, Franke WW, Moll R. Patterns of expression of trichocytic and
epithelial cytokeratins in mammalian tissues. Differentiation 1988; 39: 167–184.
5. Zaias N. Embryology of the human nail. Arch Dermatol 1963; 87:37–53.
6. Hashimoto K, Gross BG, Nelson R, Lever WF. The ultrastructure of the skin of
human embryos. III. The formation of the nail in 16–18 week old embryos. J lnvest
Dermatol 1966; 47:205–207.
7. Zaias N, Alvarez J. The formation of the primate nail plate. An autoradiographic
study in the squirrel monkey. J Invest Dermatol 1968; 51:120–136.
8. Johnson M, Comaish JS, Shuster S. Nail is produced by the normal nail bed: a
controversy resolved. Br J Dermatol 1991; 125:27–29.
9. Baden HP, Kubilus J. A comparative study of the immunologic properties of hoof
and nail fibrous proteins. J Invest Dermatol 1984; 83:327–331.
10. Heid HW, Moll I, Franke W W. Patterns of trichocytic and epithelial cytokeratins
in mammalian tissues. II. Concomitant and mutually exclusive synthesis of
trichocytic and epithelial cytokeratins in diverse human and bovine tissues.
Differentiation 1988; 37:215–230.
11. de Berker D, Leigh I, Wojnarowska F. Patterns of keratin expression in the nail
unit: an indicator of regional matrix differentiation. Br J Dermatol 1992; 127:423.
12. Westgate GE, Tidman N, de Berker D, Blount MA, Philpott MP, Leigh IM.
Characterisation of LH Tric 1, a new monospecific monoclonal antibody to the hair
keratin Ha 1. Br J Dermatol 1997; 137:24–31.
13. Moll I, Moll R. Merkel cells in ontogenesis of human nails. Arch of Dermatol Res
1993; 285:366–371.
14. Cameli N, Ortonne JP, Picardo M, Peluso AM, Tosti A. Distribution of Merkel
cells in adult human nail matrix (letter). Br J Dermatol 1998; 139:541.
15. Sirota L, Straussberg R, Fishman P, et al. X-ray microanalysis of the finger-nails in
term and preterm infants. Pediatr Dermatol 1988; 5:184–186.
16. de Berker D, Angus B. Proliferative compartments in the normal nail unit. Br J
Dermatol 1996; 135:555–559.
17. Norton LA. Incorporation of thymidine-methyl-3H and glycine-2–3H in the nail
matrix and bed of humans. J Invest Dermatol 1971; 56:61–68.
18. Kligman A. Why do nails grow out instead of up? Arch Dermatol 1961; 84: 181–
183.
19. Forslind B, Lindstrom B, Philipson B. Quantitative microradiography of normal
human nail. Acta Dermatol Venereol (stockh) 1971; 51:89–92.
20. Hashimoto K. Ultrastructure of the human toenail. I. Proximal nail matrix. J Invest
Dermatol 1971; 56:235–246.
21. Kitahara T, Ogawa H. Coexpression of keratins characteristic of skin and nail
differentiation in nail cells. J Invest Dermatol 1993; 100:171–175.
22. Lynch MH, O’Guin WM, Hardy C et al. Acid and basic hair/nail (‘hard’) keratins:
their co-localisation in upper corticle and cuticle cells of the human hair folicle and
their relationship to’soft’ keratins. J Cell Biol 1986; 103:2593– 2606.
23. Lacour JP, Dubois D, Pisani A, Ortonne JP. Anatomical mapping of Merkel cells in
normal human adult epidermis. Br J Dermatol 1991; 125:535–542.
24. de Berker D, Wojnarowska F, Sviland L, et al. Keratin expression in the normal
nail unit: markers of regional differentiation. Br J Dermatol 2000; 142:89–96.
25. Sinclair RD, Wojnarowska F, Dawber RPR. The basement membrane zone of the
nail. Br J Dermatol 1994; 131:499–505.
26. Haneke E. The human nail matrix: a flow cytometric and immuunohistochemical
studies. In: Clinical Dermatology in the Year 2000. Book of Abstracts, May: 1990.
27. Purkis FE, Steel, JB, Mackenzie IC et al. Antibody markers of basal cells in
complex epithelia. J Cell Sci 1990; 97:39–50.
28. de Berker D, Sviland L, Angus BA. Suprabasal keratin expression in the nail bed: a
marker of dystrophic nail differentiation. Br J Dermatol 1995; 133 (suppl. 45): 16.
29. Fanti PA, Tosti A, Cameli N, Varotti C. Nail matrix hypergranulosis. Am J
Dermatopathol 1994; 16:607–610.
30. Kitahara T, Ogawa, H. Variation of differentiation in nail and bovine hoof cells. J
31. Kitahara T, Ogawa H. Cellular features of differentiation in the nail. Microsc Res
Techniques 1997; 38:436–442.
32. Picardo M, Tosti A, Marchese, et al. C. Characterisation of cultured nail matrix

cells. J Am Acad Dermatol 1994; 30:434–440.
33. Nagae H, Nakanishi H, Urano Y, Arase S. Serial cultivation of human nail matrix
cells under serum-free conditions. J Dermatol 1995; 22:560–566.
34. Boot PM, Rowden G, Walsh N. The distribution of Merkel cells in human fetal and
adult skin. Am J Dermatopathol 1992; 14:391–396.
35. Munro CS, Carter S, Bryce, C. et al. A gene for pachyonychia congenita is closely
linked with the gene cluster on 17ql2-q21. J Med Genet 1994; 31: 675–678.
36. McLean WHI, Rugg EL, Lung, DP et al. Keratin 16 and 17 mutations cause
pachyonychia congenita. Nat Genet 1995; 9:273–278.
37. Corden LD, McLean WH. Human keratin disease: hereditary fragility of specific
epithelial tissues. Exp Dermatol 1996; 5:297–307.
38. Hohl D. Steatocystoma multiplex and oligosymptomatic pachyonychia congenita
of the Jackson-Lawler type. Dermatology 1997; 195:86–88.
39. Covello SP, Smith FJD, Sillevis Smitt JH et al. Keratin 17 mutations cause either
steatocystoma multiplex or pachyonychia congenita type 2. Br J Dermatol 1998;
139:475–480.
40. Smith FJ, Jonkman MF, van Goor H. et al. A mutation in human keratin K6b
produces a phenocopy of the K17 disorder pachyonychia congenita type 2. Hum
Mol Genet 1998; 7:1143–1148.
41. Bowden PE, Haley JL, Kansky, A. et al. Mutation of a type II keratin gene (K6a) in
pachyonychia congenita. Nat Genet 1995; 10:363–365.
42. Swensson O, Langbein L, McMillan, JR. et al. Specialised keratin expression
pattern in human ridged skin as an adaptation to high physical stress. Br J Dermatol
1998; 139:767–775.
43. Baden H. Common transglutaminase substrates shared by hair, epidermis and nail
and their function. J Dermatol Sci 1994; 7 (suppl.): S20-S26.
44. Manabe M, O’Guin WM. Existence of trichohyalin-keratohyalin hybrid granules:
co-localisation of 2 major intermediate filament-associated proteins in non-
follicular epithelia. Differentiation 1994; 58: 65–75.
45. O’Keefe EJ, Hamilton EH, Lee SC, Steiner P. Trichohyalin: a structural protein of
hair, tongue, nail and epidermis. J Invest Dermatol 1993; 101:65s-71s.
46. Lavker RM, Risse B, Brown, et al. H. Localisation of plasminogen activator
inhibitor type 2 (PAI-2) in hair and nail: implications for terminal differentiation. J
47. Astbury WT, Sissons WA. X-ray studies of the structure of air, wool and related
fibres. III. The configuration of the keratin molecule and its orientation in the
biological cell. Proc. Roy. Soc. Lond A. 1935; 150:533.
48. Derksen JC, Herringa GC, Weidinger A Acta Neerl Morphol 1937; 1:31–37.
49. Baden HP. The physical properties of nail. J. Invest. Dermatol. 1970; 55: 115–122.
50. Forslind B. Biophysical studies of the normal nail. Acta Dermato Venereol (Stockh)
1970; 50:161–168.
51. de Berker DAR, MaWhinney B, Sviland L. Quantification of regional matrix nail
production. Bri J Dermatol 1996; 134:1083–1086.
52. Samman P. The ventral nail. Arch Dermatol 1961; 84:192–195.
53. Johnson M, Schuster S. Continuous formation of nail along the nail bed. Br J
Dermatol 1993; 128:277–280.
54. Johnson M, Comaish JS, Shuster S. Nail is produced by the normal nail
bed:acontroversyresolved. Br J Dermatol 1991; 125:27–28.
55. Finlay AY, Moseley H, Duggan TC. Ultrasound transmission time: an in vivo
guide to nail thickness. Br J Dermatol 1987; 117:765–770.
56. Jamec GBE Serup J. Ultrasound structure of the human nail plate. Arch Dermatol
1989; 125:643–646.
57. Hamilton JB, Terada H, Mestler GE. Studies of growth throughout the lifespan in
Japanese: growth and size of nails and their relationship to age, sex
heredityandotherfactors. J. Gerontol. 1955; 10:401–415.
58. Hillman RW. Fingernail growth in the human subject. Hum Biol. 1955; 27: 274–
283.
59. Bean WB.Thirty five years of observation. Arch. Intern. Med. 1980; 140:73–76.
60. Bean WB. A note on fingernail growth. J Invest Dermatol 1953; 20:27–31.
61. Dawber R. Fingernail growth in normal and psoriatic patients. Br. J. Dermatol.
1970; 82:454–457.
62. Samman PD In: The Nails in Disease, 2nd ed.: London: William Heinemann
Medical Book, 82.
63. Zaias N.The movement of the nail bed. J Invest Dermatol 1967; 48:402–403.
64. Jarrett A, Spearman RC. Histochemistry of the human nail. Arch. Dermatol. 1966;
94:652–657.
65. Maibach MI, Epstein WL. Dynamics of finger nail formation: cysteine-35S
Incorporation. Clin Res. 1966; 14:270.
66. Jalili MA, Al Kassab S Lancet 1959; 2:108–110.
67. Pascher G. Bestandteile der menschlichen Hornschicht. Quantitative Skleroprotein-
Bausteinanalysen. Arch. Klin. Exp. Dermatol 1964; 218: 111–125.
68. Crounse RG In: Lyne-Short Biology of the Skin and Hair Growth, 1965; 218: p.
307.
69. Price VH, Odom RB, Ward WH, Jones FT. Trichothiodystrophy: sulfurdeficient
brittle hair as a marker for a neuroectodermal symptom complex. Arch Dermatol.
1980; 116:1375–1384.
70. Jacobs A. The effect of iron deficiency on the tissues. Gerontol Clin. 1971; 13:61–
68.
71. Young RW, Newman SB, Capott RJ. Strength of fingernails. J. Invest. Dermatol.
1965; 44:358–360.
72. Newman S.B, Young R.W. Indentation hardness of the nail. J. Invest. Dermatol.
1967; 49:103–105.
73. Robson JR, el-Tahawi HD. Hardness of human nail as an index of nutritional
status: apreliminary communication. Br J Nutr. 1971; 23:1272–6.
74. Forslind B, Nordström G, Toyer D, Eriksson K. The rigidity of human finger-nails:
a biophysical investigation on influencing physica parameters. Acta Dermatol
Vernereol (Stockh) 1980; 60:217–222.
75. Vellar OD. Composition of human nail substance. Am J Clin Nutr 1970; 23: 1272–
4.
76. Spruit D. Measurement of water vapour loss through human nail in vivo. J Invest.
Dermatol. 1971; 56:359–361.
77. Nilsson GE. Measurement of water exchange through skin. Med Biol Eng Comput
1977; 15:209–218.
78. Lindström B. 1955, Acta Radiol. (Stockh) suppl. 125 (thesis).
79. Forslind B, Lindström B, Forslind B. Pachyonychia congenita. A histologic and

microradiographic study. Acta Dermat Venereol (Stockh) 1971; 51:89–92.
80. Forslind B, Wroblewski R, Afzelius BA. Calcium and sulphur location in normal
nail. J. Invest. Dermatol. 1976; 67:273–275.
81. Forslind B, Thyresson N. On the strustures of the normal nail. Arch. Dermatol.
Forsch. 1975; 251:199–204.
82. Harrison WW, Clementa GG. Survey analysis of trace elements in human fin-
gernails by spark source mass spectrometry. Clin. Chim. Acta 1972; 36:485–492.
83. Oimomi M, MaedaY, Hata F. et al. Glycosylation levels of nail proteins in diabetic
patients with retinopathy and neuropathy. Kobe J Med Sci 1976; 67: 273–275.
84. Sueki H, Nozaki S, Fujisawa, R. et al. Glycosylated proteins of skin, nail and hair:
application as an index for long-term control of diabetes mellitus. J Dermatol 1989;
16:103–110.
85. Djaldetti M, Fishman P, Harpaz D, Lurie B. X-ray microanalysis of the fingernails
in cirrhotic patients. Dermatologica 1987; 174:114–116.
86. MatsumotoT, Sakura N, Ueda K. Steroid sulphatase activity in nails: screening for
X-linked ichthyosis. Pediatr Dermatol 1990; 7:266–269.
87. Serizawa S, Nagai T, Ito M, Sato Y. Cholesterol sulphate levels in the hair and
nails of patients with recessive X-linked ichthyosis. Clin Exp Dermatol 1990; 15:
13–15.
88. Salamon T, Lazovic-Tepavac O, Nikulin, A. et al. Sudan IV positive material of the
nail plate related to plasma triglycerides. Dermatologica 1988; 176: 52–54.
89. Behne D, Gessner H, Kyriakopoulos A. Information on the selenium status of
several body compartments of rats from the selenium concentrations in blood
fractions, hair and nails. J Trace Elem Med Biol 1996; 10:174–179.
90. Rogers M, Thomas DB, Davis, S. et al. A case control study of oral cancer and pre-
diagnostic concentrations of selenium and zinc in nail tissue. Int J Cancer 1991; 48:
182–188.
91. van den Brandt PA, Goldbohm RA, vant Veer P. et al. Toenail selenium levels and
the risk of breast cancer. Am J Epidemiol 1994; 140:20–26.
92. Yoshizawa K, Willett WC, Morris, SJ. et al. Study of the prediagnostic selenium
levels in toenails and the risk of advanced prostate cancer. J Nat Cancer Inst 1998;
90:1219–1224.
93. Garland M, Morris JS, Colditz, GA et al. Toenail trace element levels and breast
cancer: aprospective study. Am J Epidemiol 1996; 144:653–660.
94. Vannucchi HF, Varo R.M, Cunha DF, Marchini JS. Assessment of zinc nutritional
status ofpellagrapatients. Alcohol Alcohol 1995; 30:297–302.
95. Kaneshige T, Takagi K, Nakamura, S. et al. Genetic analysis using fingernail
DNA. Nucleic Acid Res 1992; 20:5489–5490.
96. Tahir M, Watson N. Typing of DNA HLA-DQa alleles extracted from human nail
material using polymerase chain reaction. J Forensic Sci CA 1995; 40: 634–636.
97. Wilhelm M, Hafner D, Lombeckl, Ohnesorge FK. Monitoring of cadmium, copper,
lead and zinc status in young children using toenails: comparison with scalp hair.
Sci Total Environ 1991; 103:199–207.
98. Das D, Chatterjee A, Badal, K. et al. Arsenic in ground water in six districts of
West Bengal, India: the biggest arsenic calamity in the world. Analyst 1995; 120:
917–924.
99. Harrington J, Middaugh J, Housworth J. A survey of a population exposed to high

concentrations of arsenic in well water in Fairbanks, Alaska. Am J Epidemiol
1978; 108:377–385.
100. Karagas MR, Morris JS, Weiss JE. Toenail samples as an indicator of drinking
water arsenic exposure. Cancer Epidemiol, Biomarkers Prev 1996; 5:849– 852.
101. Nowak B. Occurrence of heavy metals, sodium, calcium and potassium in human
hair, teeth and nails. Biol Trace ElemRes 1996; 52:11–22.
102. Gamelgaard B, Anderson JR. Determination of nickel in human nails by adsorption
differential-pulse voltametry. Analyst 1985; 110:1197–1199.
103. Suzuki O, Hattori H, Asano M. Nails as useful materials for detection of
methamphetamine or amphetamine abuse. Forensic Sci Inter 1984; 24: 9–16.
104. Suzuki S, Inoue T, Hori H, Inayama S. Analysis of methamphetamine in hair, nail,
sweat and saliva by mass fragmentography. J AnalToxicol 1996; 144: 653–660.
105. Miller M, Martz R, Donnelly B. (1994). Drugs in keratin samples from hair,
fingernails and toenails. In: Second International Meeting on Clinical and Forensic
Aspect of Hair Analysis (abstr), Genoa, Italy, June 6–8, p. 39.
106. Cirimele V, Kintz P, Mangin P. Detection of amphet-amines in fingernails: an
alternative to hair analysis. Archives of Toxicology 1995; 70:68–69.
107. Skopp G, Pötsch L. A case report on drug screening of nail clippings to detect
prenatal drug exposure. Ther Drug Monit 1997; 19:386–389.
108. Dykes PJ,Thomas R, Finlay AY. Determination of terbinafine in nail samples
during treatment for onychomycoses. Br J Dermatol 1990; 123:481–486.
109. Matthieu L, de Doncker P, Cauwenburgh, G. et al. Itraconazole penetrates the nail
via the nail matrix and the nail bed: an investigation in onychomycosis. Clin Exp
Dermatol 1991; 16:374–376.
110. Munro CS, Shuster S. The route of rapid access of drugs to the distal nail plate.
Acta Dermatol Venereol (Stockh) 1992; 72:387–388.
111. Rashid A, Scott EM, Richardson MD. Inhibitory effect of terbinafine on the
invasion of nails by Trichophyton mentagrophytes. J Am Acad Dermatol 1995; 33:
718–723.
112. Ofori-Adjei, D. Ericsson, O. (1985) Chloroquine in nail clippings. 2 (8450), 331.
About the Editors
BO FORSLIND was Professor, Department of Medjcal Biophysics and

Biochemistry, Karolinska Institutet, Stockholm, Sweden. The recipient of the
Research Award (1998) from the Swedish Allergy and Cancer Foundation, he
received the Ph.D. degree (1970) from the Karolinska Institutet, Stockholm,
Sweden.
MAGNUS LINDBERG is Professor of Occupational Dermatology,
Department of Medicine, Karolinska Institutet, Stockholm, Sweden, and Senior
Consultant in Occupational and Environmental Dermatology, Center of Public
Health, Stockholm County Council, Sweden. He received the Ph.D. degree
(1982) from the Karolinska Institutet, Stockholm, Sweden.
Index
Acylceramide, 81 Dundee experimental bald rat, 369

Adhesion molecules Alopecia areata, pathology, 367, 368
alopecia areata, 368 Amino acid analysis (AAA), 263
irritant contact dermatitis, 232 Amniotes skin barrier, 179
Aging and melanocytes, 316 Amphetamine in nails, 424
Air-water lipid interface, 37 Amphibian skin barrier, 176
A-layer, 257, 266, 273 Anagen-catagen transition, 313
Albinism, 305, 328 Anagen in hair growth, 251, 336
Alopecia areata, Analytic techniques
adhesion molecules, 368 domain formation, 42, 43
autoimmune diseases, 358, 359, 366 lipid bilayers, 40
cytokines, 363, 368 stratum corneum lipid, 28
Down’s syndrome, 358, 364 stratum corneum lipid, organization,
environmental factors, 358 128–129
epidemiology, 359 Androgens
genetics, 362–366 alopecia, 339, 342
[Alopecia areata] diferences in response, 343
infection, 361 hair growth, 4, 342, 336–357
inflammation, 368 insuffiency, 342
inheritance model, 358 mechanisms, 345
interleukin 1 (IL-1), 363 mode of action, 347–348
pernicious anemia, 359 receptors, 345, 350
(see also Disorders of hair pigmentary target cells, 345
unit) Animal models for alopecia areata, 369,
stress, 361 370
T-cell inflammation, 358 Antiandrogenic drugs and hair growth, 343
thyroid disorders, 359 Apoptosis
TNF-a, 364 hair fiber, growth, 259
vitiligo, 359 hair pigmentary unit, 297
Alopecia areata, genetics keratinization, 2
chromosome 18, 364 melanocytes, hair, 314, 317
major histocompatibility complex Aquatic adaptation and skin barrier, 186
(HLA), 362 Aquatic mammals, 187
Alopecia areata, models of Arsenic poisoning and nails, 424
C3H/HeJ mouse, 370 Atomic force microscopy (AFM)
hair research, 263, 271
435
436 INDEX
lipid, 28 Cadherins, 298

stratum corneum lipid, 40, 42, 43 Calcium content
Atopic dermatitis melanocytes, hair, 323
ceramides, 117 nail composition, 415, 418, 419
irritant contact dermatitis, 200, 217 Calorimetry
lamellar bodies/granules, 120 lipid phase transitions, analysis of, 31
skin penetration, 197, 200 lipid, 28
[Alopecia dermatitis] phase tranformations, 31
stratum corneum function, 117, 119 Canities in hair, 318
waterholding capacity, 200 Catagen in hair growth, 251, 336
xerosis, 200 Catagen and melanocytes, 314
Attenuated total reflectance FTIR, 66 Cell membrane complex in cuticle cells,
Autoimmune diseases and alopecia areata, 275–280
358, 359, 366 Ceramides
Avians atopic dermatitis, 117
permeability, 184 cer 1-cer 6, 34
skin barrier, 184 domain formation, 40, 41
Axis of the nail plate, 382 FTIR spectroscopy, 67
HPTLC, 97
Barrier-forming process, stratum corneum isolated and phase behavior, 120–122
models, 0 –154 keratinization, lipid, 2
Barrier function, 5, 96 mixtures, 123
(see also Skin barrier) spingosides, 97
Barrier models, st. corneum, 57 stratum corneum lipid, 13, 20, 32, 34,
Barrier properties 75, 81, 97
moisturizers, 202 -hydroxy acids, 97
percutaneous absorption, 200 protonated in FTIR. spectroscopy, 66
solvents, 204 Cetacean
stratum corneum, 21 cornefied cell envelope, 189
stratum corneum lipids, 43 epidermis, 188
Basal membrane in nails, 400 keratin, 191
Basal membrane in skin, 9, 10 permeability, 189
Benzalkonium chloride, 222, 230–239 Chain melting and lipid phases, 30
Bilayer configuration Chemical potential in st. corneum, 47
lipid aggregation, 22 Chiral dppc and lipid domains, 40
stratum corneum lipids, 43 Cholesterol
Biochemical techniques in hair research, domain formation, 40, 41
263 stratum corneum lipids, 20, 32, 34, 75,
Bioengineering techniques 82, 97, 98
erythema measurements, 219 stratum corneum lipids, mixtures, 123
evaporimetry, 219 stratum corneum models, 156
guidelines, 219 Cholesterol esters, 13, 82
irritant contact dermatitis, 219 Cholesterol sulfate
laser doppler flowmetry, 219 ichthyosis, 202
Biological membranes and lipids, 39, 43 stratum corneum lipids, 97
Brewster angle microscopy, 40 stratum corneum lipids, mixtures, 123
Bricks and mortar model, 14, 20 Chromosome 21 and alopecia areata, 364
Circumscribed poliosis, 327
INDEX 437
Claws, 5 [Cuticle cell]

Clinics of irritant contact dermatitis, 215 scanning probe microscopy, 271
Cocaine in nails, 424 SSIMS, 270
Colloidal forces analysis, 29 ultrastructure, 264
Conceptual models of st. corneum, 139 X-ray photoelectron spectroscopy, 270
Conformational changes, st. corneum cell envelope, 270, 272, 273
lipids, 148 Cuticle (eponychium) in nail, 377
Consolidation in hair growth, 251 Cuticle in hair, 251, 256, 261
Contact dermatitis, 213 Cuticle hair fiber
cosmetics and skin care products, 215 A-layer, 257, 266
etiology, 213, 216 cell membrane complex, 275–280
Cornefied cell envelope, 175 cuticle cells, 263
cetacean, 189 differentiation of, 280
corneocytes, 11 electron-dense granules, 280
ichthyosis, 120 endocuticle, 257, 266
keratinization, 2 epicuticle, 266
stratum corneum, 75, 131 (see also Fiber cuticle surface
stratum corneum lipids, 82 membrane)
Corneocytes, 8 exocuticle, 257, 273
corneocyte envelope, 11 fiber cuticle surface membrane, 266
-( -glutamyl) lysine isopeptide bonds, isolation of, 275
11 lipids, 257
fillagrin, 10 mechanical properties, 257
proteins, 11 size, 256
stratum corneum, structure, 96, 113 structure, ultrastructure, 264
swelling, 5 surface, 261, 264
-hydroxy ceramides, 11 weathering, 257
Corneodesmosomes, 5, 131 Cysteine
Cortex exocuticle, 273, 274
hair fiber, 2, 3, 251, 261 keratinization, 1
intermediate filaments (IFs), 257 Cystic fibrosis and nails, 423
keratin filaments , 257 Cytokines
-Keratin, 257 alopecia areata, 363, 368
Cosmetics, stratum corneum, 9 irritant contact dermatitis, 219, 225,
Cosmetics and skin care products, 215 231
Croton oil, 230
Crystalline packing Delipidization of solvents, 204
lipid phase, 147 Dermal factors for hair growth, 347–348
lipids, aggregation, 22 Dermal papilla in hair follicle, 250, 251,
NMR, 60 252
C3H/HeJ mouse, 370 Dermal papilla hypothesis (model) for hair
Cubic phase of lipids, 27 growth, 347–348, 350, 353
Cubiclike membrane, st. corneum lipids, Desmosomes, 2, 10
146 Desquamation of st. corneum, 96, 159
Cuticle cell Detergents and nail rigidity, 422
cuticle, hair fiber, 263 Detergents and percutaneous absorption,
gel electrophoresis (PAGE), 274 199
438 INDEX
Development of hair color, 296 Eicosanoids and irritancy, 232

Diabetes and nails, 422 Elastic modulus in nails, 422
Differential scanning calorimetry (DSC) Electrolytes, lipid phase behavior, 36, 419
lipids, 28 Electron-dense granules in cuticle cells,
stratum corneum models, 69 280
Differentiation Electron diffraction (TEM) and st. corneum
cuticle cells, hair fiber, 280 lipids, 104, 105, 113, 115
hair, 252 Electrostatic forces and lipids, 99
nail, 381 Elemental content and irritancy, 220
(see also Keratinization) Endocuticle, 257, 266, 274–275
Diffraction curves at corneum, 110 Endoplasmic reticulum and lamellar
Diffusion barrier bodies, 9
epidermis, function, 9 Environment
pathways, 44 alopecia areata, 358
stratum corneum lipid, 43 nail composition, 423
Dihydrotestosterone (see Hair growth) Epicuticle, 266
Disease and skin penetration, 197 Epidemiology
Disease and st. corneum, 117, 130 alopecia areata, 359
Distal groove in nail, 378 hand eczema, 213
Dithranol, 220 Epidermal cyst and lipids, 81
DNA in nails, forensic applications, 423 Epidermal melanocytes, 302
Dolphin skin, 173 Epidermis
Domain formation cetacean, 188
analysis, techniques used, 42, 43 desmosomes, 10
chiral dppc, 40 hemidesmosomes, 10
fatty acids, ceramides, cholesterol, 40, hippopotamus, 187
41 lamellar bodies, 7
lipid bilayers, 39 membrane-coating granules, 7
lipid composition, 40 skin, structure, 5
lipid monolyers, 39 stem cells, 9
lipid phase diagrams, 40 whales, 188
membrane permeability, 45 Epidermis function, 9
shape and size of, 40 Epidermis structure, 9
stratum corneum lipid, 44 Eponychium, 377
thermodynamic variables, 40 Erythema measurements, 219
Domain mosaic model E-selectin and irritancy, 232
lipid phases, 22 Etiology
mechanical properties, 22 contact dermatitis, 213, 216
stratum corneum models, 5, 14, 22, 76 irritant contact dermatitis, 216
transport properties, 22 Eumelanosome, 304
Domains and st. corneum lipids, 21 Evaporimetry, 219
DOPA in melanin, 300 Evolutionary selection in hair color, 295
Dorsal nail plate, 4 Evolution of the skin barrier, 171
Down’s syndrome and alopecia areata, Exocuticle
358, 364 A-layer, 273
Dry mass and sulfur in nails, 417 cuticle, hair fiber, 257, 273
Dundee experimental bald rat (DEBR), cysteine, 273, 274
369 sulfur content, 274
INDEX 439
Exocytosis of lamellar bodies, 9 fiber cuticle suface membrane, 281

Exogenous ingested substances in nails, hair pigmentary unit, 297, 310
423 skin barrier, 10
Experimental models hair growth, 337, stratum corneum, 141–143, 174
350 Fossorial adaptation
skin barrier, 182
Fast field echo microscopy (FFEM), 120 Fourier transform infrared spectroscopy
Fatty acids (FTIR)
domain formation, 40, 41 attenuated total reflectance, 66
fiber cuticle surface membrane, lipid, ceramide, protonated, 66
269 ceramides, 67
FTIR spectroscopy, 67 fatty acids, 67
K, 36 infrared reflection-absorption
stratum corneum lipids, 13, 20, 32, 34, spectroscopy, 66
75, 82, 97, 98 model membranes, 65
X-ray diffraction, 39 palmitic acid, deuterated, 66
Feathers, 173 stratum corneum lipid, 57, 65–68
Fiber cuticle suface membrane stratum corneum lipid phases, 115
formation of, 281 stratum corneum models, 66, 67
maple syrup urine disease, 284 stratum corneum, structure, 113
mutations, inherited defects, 284 Freeze fracture SEM and lipids, 28
epicuticle, 266 Frog skin, 176
FCUSM, 267 Function of
investigative techniques, models, 270, hair color (pigmentation), 295
271 hair growth, 339, 339
lipids in, 268, 269 skin barrier, 171
proteinaceous component, 268 Fur, 173
proteins, 272
structure of, 266 Gas liquid chromatography (GLC), 79
Fiber cuticle surface membrane, lipids Gel electrophoresis (PAGE)
fatty acids, 269 cuticle cell, 274
F-layer, 269 hair research, 263
18-methyleicosanoic acid, 269, 270, Gel phase and lipid aggregation, 22
270 Genetic poliosis, 328
Fiber cuticle surface membrane, proteins Genetics of alopecia areata, 362–366
involucrin, 272 GLC-electrospray ionization (GLC-ESI),
protein matrix, 272 79
Fick’s first law, 199 GLC-flame ionization detector (GLC-
Filaggrin FID), 79
corneocytes, 10 GLC-mass spectrometry (GLC-MS), 79
histidine, 10 -( -Glutamyl) lysine isopeptide bonds, 11
keratinization, 2 Grain boundaries of st. corneum lipids,
nail proteins, 400 130
F-layer, 269 Growth dynamics of nails, 0 , 400
Fluorescence quenching, 40 Guidelines for bioengineering techniques,
Fluorescences microscopy, 40 219
Formation of
440 INDEX
Hair bulb inflammation in alopecia areata, degenerative changes, apoptosis, 323

367, 368 general health, 324
Hair bulb structure, 251 hair colorants, 324
Hair color hair pigmentary unit, 318
biology of, 293–335 melanocytes, 318
development of, 296 osteopenia, 324
evolutionary selection, 295 pathogenesis of, 319, 325
function of, 295 senile white hair, 324
heavy metals, binding of, 296 smoking, 324
melanocortin-1 receptor, 296 structure, 323
UVR, 296 Hair growth, 3
Hair colorants and gray hair, 324 adaptation of, 337
Hair disorders and hair color, 326–328 alopecia areata, pathology, 367
Hair fiber anagen, 251, 336
cortex, 2, 3 androgen influence, 4, 336–357
differentiation, 252 catagen, 251, 336
hair follicle, structure of, 250 consolidation, 251
inner root sheath, 3 dermal factors, pp. 347–348
medulla, 3 differentiation, 252
shape of, 252 function of, 339, 339
wool, references, 263 hair follicle in, 336
Hair fiber growth and apoptosis, 259 keratinization, 251
Hair fiber structure sex differences, 339
cortex, 251, 261 telogen, 336
cuticle, 251, 256, 261 variations of, seasonal, 338
dermal papilla, 252 Hair growth control
inner root sheath, 251, 252 experimental approaches, 337
medulla, 251, 261 hair follicles, 250, 343
outer root sheath, 252 hormones, 337
root sheath, 252 Hair growth disorders
surface, 261 alopecia, 339
Hair fiber studies, methods, 263 (see also Alopecia areata)
Hair follicle androgenetic alopecia, 339, 342
hair growth, 336 hirsutism, 339, 343
hair growth control, 250, 343 [Hair growth disorders]
[Hair follicle] male pattern baldness, 4, 339
investigative techniques, 251 Hair growth hormones (see Androgens)
structure and function, 3, 251 Hair growth models
alopecia areata, 367, 368 androgen receptor in, 350
Hair follicle pigmentary unit (see Hair dermal papilla hypothesis (model),
pigmentary unit) 347–348, 350, 353
Hair follicle structure experimental model, 350
dermal pailla, 250, 251 mitogenic factors, 350, 352
hair bulb, 251 paracrine factors in, 350
hair fiber, 250 testosterone metabolism in, 350
matrix, 251, 252 Hair keratins
Hair graying and -keratin, -keratin, 254
INDEX 441
intermediate filaments (IFs), 254 Histidine and filaggrin, 10

sulfur content, 254 Histopathology and irritant contact
Hair loss and alopecia areata, 367 dermatitis, 219
Hair pigmentary unit Homeostasis of skin barrier, 11
anagen-catagen transition, 313 Hormones and hair growth, 337
apoptosis, 297 Hormones and melanocytes, 309
cadherins, 298 HPTLC and ceramides, 97
disorders of, 326–328 Huxley layer, 254
formation of, 297, 310 Hydrogen bondings and lipids, 99
hair graying, 318 Hydrophobic vs. hydrophilic pathways, 16
integrins, 298 Hydrophobic interactions and lipids, 99
matrix, 298 -Hydroxy acids
melanocytes, 297, 298, 302 ceramides, 97
mutations, 297 stratum corneum lipid, 75, 81
oncogenes, 298 -Hydroxy ceramides
telogen-anagen transition, 310 corneocytes, 11
Hair pigmentary unit disorders keratinization, lipid, 2
albinism, 328 stratum corneum lipid, 14, 75, 81
alopecia areata, 326 6-Hydroxysphingosine, 82
(see also Alopecia areata) Hyponychium, 378
circumscribed poliosis, 327
genetic poliosis, 328 latroscan and TLC, 78
vitiligo, 326 ICAM-1 in irritant contact dermatitis, 232
Hair research Ichthyosis
amino acid analysis, 263 cholesterol sulfate, 202
atomic force microscopy, 263, 271 corneocyte cell envelope, 120
biochemical techniques, 263 irritant contact dermatitis, 202
gel electrophoresis, 263 lamellar bodies/granules, 120
HPLC, 263 [Ichthyosis]
infrared spectroscopy, 263 skin permeability, 197, 202
scanning electron microscopy, 263 stratum corneum function, 117, 119
secondary ion mass spectroscopy, 263 Immediate skin reactions of solvents, 204
transmisson electron microscopy, 263 Immediate skin whitening of solvents, 204
[Hair research] Immunhistopathology irritant contact
X-ray photoelectron spectroscopy dermatitis, 220
(XPS), 263 Immunohistochemistry and nail keratins,
Hand eczema prevalence, 213 390
Heavy metals and hair color, 296 Immunohistochemistry and nail proteins,
Heavy metals and nails, 424 399
Hemidesmosomes, 10 Immunological defense in epidermis, 9
Henley layer, 254 Impact resistance of skin barrier, 173
High-performance liquid chromatography Inflammation
(HPLC) alopecia areata, 368
hair research, 263 epidermis, function, 9
stratum corneum lipids, 77, 78 irritant contact dermatitis, 217
Hippopotamus skin barrier, 173, 174, 187 Infrared reflection-absorption spectroscopy
Hirsutism, 339, 343 (IRRAS), 66
442 INDEX
Infrared spectroscopy (IR) in hair research, histopathology, 219

263 ICAM-1, 232
Inheritance model of alopecia areata, 358 ichthyosis, 202
Inner root sheath (IRS) immunhistopathology, 220
hair fiber, structure, 3, 251, 252 inflammation, 217
Henley layer, 254 interferon- , 232
Huxley layer, 254 interindividual variations, 216
cuticle, 255 interleukin-1 , 225
Integrins in hair, 298 investigative techniques, 218
Interactions of irritants, 218 Langerhans cells, 225
Intercellular lipids st. corneum, 5, 7, 96 LFA-1, 232
Interferon- (IFN- ), 232 noninvasive techniques, 219
Interindividual variations and irritancy, oxidative stress, 232
216 particle probes, 220
Interleukin-1 proliferation, 235
irritant contact dermatitis, 225 repeated exposure, 235, 236
alopecia areata, 363 skin barrier function, 217
Intermediate filaments (IFs) solvents, 204, 206
cortex, hair, 257 TEWL, 219
hair keratin, 254 TNF- , 225
keratinization, 1 ultrastructure, 219
Intersection-free membrane unfolding, VCAM-1, 232
143, 162 Irritants
Invertebrates skin barrier, 173 benzalkonium chloride, 222, 230–239
Investigative techniques croton oil, 230
fiber cuticle surface membrane, 270, dithranol, 220
271 interactions, 218
hair follicle, 251 mode of action, 218
irritant contact dermatitis, 218 nonanoic acid, 220, 222, 225, 230–239
In vivo gradients in st. corneum, 139 repeated applications (exposures), 220
Involucrin single exposure, pp. 219–222
fiber cuticle surface membrane, sodium lauryl sulfate, 220, 222, 225,
proteins, 272 230–239
keratinization, 2
nail proteins, 399 Keratin filaments in hair cortex, 257
Irritancy and solvents, 206 Keratin synthesis in nail, 381
Irritant contact dermatitis Keratinization
adhesion molecules, 232 apoptosis, 2
atopic dermatitis, 200, 217 ceramides, 2
bioengineering techniques, 219 cornified cell envelope, 2
clinical appearence, 215, 220 cysteine, 1
cytokines, 219, 225, 231 desmosomes, 2
definition of, 215 filaggrin, 2
eicosanoids, 232 hair growth, 251
elemental content, 220 intermediate filaments, 1
E-selectin, 232 involucrin, 2
etiological factors, 216 keratins, 1
experimenatal designs, 217
INDEX 443
loricin, 2 colloidal forces analysis, 29

oxidative stress, 235 concentration, solvent properties, 27
sulfur content, 1 crystalline packing, 22
thioester bonds, 2 cubic phase, 27
-hydroxy ceramides, 2 gel phase, 22
Keratinocyte proliferation and irritancy, lamellar bodies/granules, 27
235 lipid aggregation in water, 22
Keratinocytes, 9 lipid phases, 22
Keratins liquid crystalline phase, 29
cetacean, 191 micelles, 24
keratinization, 1 Poisson-Boltzman equation, 29
nail structure, 4 temperature, effect of, 27
stratum corneum, structure, 116 water and lipid aggregation, 22
wide-angle X-ray diffraction, 113 water, hydration force, 28
Keratins in hair (see Hair keratins) water, swelling, 24
Keratins in nails (see Nail keratins) X-ray diffraction, 22
Keratins in skin (see Skin keratins) Lipid bilayers
Keratohyaline granules, 9 analysis, techniques used, 40
Ki67 and proliferation, 235 biological membranes, 39
domain formation, 39
Lamellar bodies, 174, 189 lipid composition, 39
atopic dermatitis, 120 lipid mixtures, 39
endoplasmatic reticulum, 9 phase diagrams, 40
epidermis, 7 phase segregation, 39
epidermis, structure, 9 stratum corneum lipid, 21
exocytosis, 9 Lipid chemical properties, 32
ichthyosis, 120 Lipid composition
lipid, aggregation, 27 domain formation, 40
stratum corneum lipid, 97 lipid bilayers, 39
stratum corneum, structure, 75 stratum corneum models, 139
Lamellar granules (see Lammellar bodies) confidence intervals, stratum corneum
Landmann model, 141 lipids, 73, 81–92
Langerhans cells Lipid contamination, 80
epidermis, structure, 9 Lipid domains (see Stratum corneum lipids)
irritant contact dermatitis, 225 Lipid mixtures
Laser doppler flowmetry, 219 lipid bilayers, 39
Lateral nail fold, 377 stratum corneum lipid, models, 108–
Lateral surface pressure and lipid 109
monolayers, 37 Lipid monolayers, 37
L-DOPA in melanin, 306 air–water interface, 37
LFA-1 in irritant contact dermatitis, 232 domain formation, 39
Light microscopy and nails, 405 lateral surface pressure, 37
Light scattering detection (HPLC-LSD), lipid phases, 37
78 X-ray diffraction, 39
Linoleic acid, 75 Lipid organization
Lipid aggregation electrostatic forces, 99
bilayer configuration, 22 hydrogen-bonding, 99
hydrophobic interactions, 99
444 INDEX
micelles, 99 Lipids in stratum corneum, 11

pH, 99 Lipids of human sebum as contamination,
pressure, 99 80
stratum corneum, structure, 96, 99 Liquid crystalline phase, formation of, 29
temperature, 99 Lorenz factor, 103
van der Waals, 99 Loricin, 2
Lipid phase behavior L-phenylalanin in melanin, 306
effect of pH, 36 L-tyrosine in melanin, 306
effects of electrolytes, 36 Lunula (half-moon) in nails, 378
effects of noncharged molecules, 37
nuclear magnetic resonance, 61, 62, 65 Major histocompatibility complex (HLA),
phase diagrams, 37 362
stratum corneum lipid, 22 Male pattern baldness, 4, 339
Lipid phase diagrams domain formation, Mammals and skin barrier, 182
40 Mammals and skin permeability, 175
Lipid phases Maple syrup urine disease (MSUD), 284
chain melting, 30 Marine mammals and skin permeability,
domain mosaic model, 22 175
lipid monolyers, 37 Markers of nail growth, 400, 402
lipid, aggregation, 22 Matrix of hair, 251, 252
permeability of different, 45 Matrix and hair pigmentary unit, 298
phase behavior, varibles, 30 Measurements of nail growth, 403
phase tranformations, 30 Mechanical properties
stratum corneum lipid, 28, 43 cuticle, hair fiber, 257
stratum corneum models, 68 domain mosaic model, 22
stratum corneum, structure, 109–117 nail, 5
Lipid phase transitions stratum corneum, 5
calorimetric techniques for analysis, 31 stratum corneum, function, 160
osmotic forces, 31 Medulla
stratum corneum, structure, 115 hair fiber, 3
temperature, effect of, 31 hair fiber, structure, 251, 261
water content, 31 structure of, 257
Lipid quantitation with TLC, 77 formation of, 306
Lipid structure and membrane function, 43 L-DOPA, 306
Lipids L-phenylalanin, 306
AFM, 28 L-tyrosine, 306
calorimetry, 28 redistribution in melanocytes, 317
cuticle, hair fiber, 257 synthesis of, 306
DSC, 28 Melanin granule, 302
fiber cuticle surface membrane, 268, Melanin loading, 318
269 Melanocortin-1 receptor, 296
freeze fracture SEM, 28 Melanocyte
NMR, 28 compartments, 328
sebum, 80 epidermis, structure, 9
SAXD, 28 hair pigmentary unit, 297, 298, 302
TEM, 28 Melanocytes, hair
wide-angle X-ray diffractions, 28 aging, 316
X-ray diffraction, 24
INDEX 445
antibodies, monoclonal, 300 Mitogenic factors and hair growth, 350,

apoptosis, 314, 317 352
calcium content, 323 Model membranes
canities, 318 FTIR, 65
in catagen, disappearance of, 314 NMR, 61, 65
DOPA, 300 stratum corneum, 45, 46, 68, 71
epidermal melanocytes, relation to, Moisturizers
302 barrier properties, 202
eumelanosome, 304 [Moisturizers]
[Melanocytes, hair] natural moisturizing factors, 202
hair graying, 318 skin penetration, 202
keratinocyte interactions, 323 stratum corneum, 9
melanin granule, 302 Movements in the nail bed, 404
melanin loading, 318 Mutations
melanin, redistribution of, 317 hair pigmentary unit, 297
melanogenesis, 302, 304 fiber cuticle suface membrane, 284
melanosome, 304
pheomelanosome, 305 Nail
pigment production, 301 claws, 5
Melanogenesis differentiation, 381
endogenous factors, 308 growth dynamics, 0
exogenous factors, 308 mechanical properties, 5
hormones, regulation by, 309 morphogenesis, 378–381
melanocytes, hair, 302, 304 proliferation, 0
regulation of, 308 structure-function, 375, 404
toxins, 308 Nail bed (ventral matrix, sterile matrix), 4,
Melanosome 378, 381
in albinism, 305 Nail composition
biogenesis, 305 arsehic poisoning, 424
melanin, formation of, 306 calcium, atomic absorption
melanocytes, hair, 304 spectroscopy, 418
proton pump, 306 cocaine, amphetamine, 424
tyrosinase, 304 in cystic fibrosis, 423
Membrane coating granules in diabetes, 422
epidermis, 7, 9 DNA, forensic application, 423
Membrane folding model, 76, 137, 141, dry mass and sulfur, 417
143–146 exogenous, environmental substances,
Membrane permeability 423
domain formation, 45 exogenous, ingested substances, 423
stratum corneum lipid, 43 heavy metals, binding of, 424
18-Methyleicosanoic acid (MEA), 269– nickel, 424
270 organic and metabolic constituences,
Micelles and lipid organization, 24, 99 422
Micro-Raman mapping of st. corneum quantitative microradiography, 417
lipids, 43 scanning electron microscopic studies,
Miller indices and st. corneum lipids, 106 418
selenium, 423
446 INDEX
sulfur and calcium, 415 keratin, 4

water content, 416 lateral nail fold, 377
in X-linked ichthyosis, 423 light microscopy, 405
X-ray microanalysis, 419 lunula (half-moon), 378
Nail growth nail bed, 4, 378, 381
in disease, 402 nail matrix (nail root), 377, 381
growth dynamics, 400 nail plate, 377, 382
[Nail growth] [Nail structure]
how to measure, 403 nail surface, 4
keratin synthesis, 381 onychocorneal band, 378
macroscopic observations, 403 proximal (posterior) nail fold, 377
markers of, 400, 402 TEM, 411
measurements of, 403 ventral nail plate, 4
movements in the nail bed, 404 X-ray diffraction, 405
nail matrix (nail root), 402 X-ray diffraction and TEM, 412–415
nail plate, 402 Nail studies, methodology, 0
speed of, 403 Nail surface, 4
Nail hardness, 415 Naked mole rat, 182
Nail keratins Natural moisturizing factors (NMF), 202
immunohistochemistry of, 390 Nickel in nails, 424
organization of, 400 Nonanoic acid (NAA), 220, 222, 225, 230–
Nail matrix (nail root), 377, 381, 402 239
Nail plate Noncharged molecules in lipid
nail growth, 402 membranes, 37
nail structure, 377, 382 Noninvasive techniques, 219
sulfur, calcium, electrolytes, 419 Nuclear magnetic resonance (NMR)
water, 416 crystalline packing, 60
Nail proteins lipid phase behavior, 61, 62, 65
filaggrin, 400 lipids, 28
immunohistochemistry of, 399 membrane lipid, 61
involucrin, 399 model membranes, 61, 65
pancornulin, 399 Pake doublet, 60
sciellin, 399 stratum corneum lipid, 28, 43, 57
trichohyalin, 400 stratum corneum models, 59–65, 213
Nail rigidity, 417, 418
determination of, 420–422 Odland bodies, 9
effects of detergents, solvents, oils, Oils and nail rigidity, 422
422 Oils and percutaneous absorption, 199
elastic modulus, 422 Oncogenes and hair pigmentary unit, 298
Nails in disease, 402 Onychocorneal band, 378
Nail structure Organic and metabolic constituences in
axis of the nail plate, 382 nails, 422
basement membrane, 400 Organic solvents and percutaneous
cuticle (eponychium), 377 absorption, 199
distal groove (limiting furrow), 378 Osmotic forces in lipid phase transitions,
dorsal nail plate, 4 31
hyponychium, 378 Osteopenia, 324
INDEX 447
Outer root sheath (ORS), 252, 255 segregation of lipid bilayers, 39

Oxidative stress stratum corneum lipid, mixtures, 122
irritant contact dermatitis, 232 Phase diagrams
keratinization, 235 lipid bilayers, 40
[Oxidative stress] lipid phase behavior, 37
ROS, 235 stratum corneum lipid, 40
superoxide dismutase, 235 Phase separation st. corneum lipids, 148
Phase tranformations
Pake doublet and NMR, 60 calorimetry, 31
Palmitic acid, deuterated, (PA-d31) and lipid phases, 30
FTIR, 66 stratum corneum lipid, 130
Pancornulin in nails, 399 Pheomelanosome, 305
Particle probes Pigment production in hair, 301
irritant contact dermatitis, 220 pK and st. corneum lipids, 36, 43
nails, 419 Poisson-Boltzman equation, 29
skin, 220 Polymorphism of st. corneum lipids, 129
X-ray microanalysis, 220 Prevalence of hand eczema, 213
Penetration enhancers, 16 Proliferation
Percutaneous absorption irritant contact dermatitis, 235
atopic dermatitis, 200 Ki67, 235
barrier properties, 200 nail, 0
detergents, 199 skin, 9
factors influencing, 199 Protein matrix of fiber cuticule, 272
Fick’s first law, 199 Proteinaceous component of fiber cuticle,
oils, 199 268
organic solvents, 199 Proteins
restrictions, 199 corneocytes, 11
skin penetration, 199 fiber cuticle surface membrane, 272
Permeability of skin stratum corneum, function, 160
avians, 184 Proton pump in melanosome, 306
cetacean, 189 Proximal (posterior) nail fold, 377
ichthyosis, 202 Psoriasis and skin penetration, 197, 202
lipid phases, 45
mammals, 175 Quantitative microradiography and nail,
marine mammals, 175 417
psoriasis, 202 Quills, 173
reptiles, 180
skin, 186 Reconstructed epidermis, 96
stratum corneum lipid, 130, 175, 199 Repeated exposure and irritants, 220, 235,
Pernicious anemia, 359 236
pH Reptiles skin barrier, 179
lipid organization, 99 Reptiles skin permeability, 180
lipid phase behavior, 36 Rhinos skin, 173
stratum corneum gradient, 47 Root sheath, 252
Phase behavior ROS in oxidative stress, 235
ceramides, isolated, 120–122 Ruthenium tetroxide staining, 110, 117
lipid phases, 30
448 INDEX
Sandwich model, 16, 100, 123–128 vertebrates, 173

Scales, 173 aquatic adaptation, 186
Scanning electron microscopic (SEM) intersection-free membrane unfolding,
hair research, 263 143, 162
nail composition, 418 Skin barrier function
Scanning probe microscopy (SPM) and formation of, 10
hair, 271 homeostasis, 11
Sciellin in nails, 399 irritant contact dermatitis, 217
Secondary ion mass spectroscopy (SIMS), TEWL, 8, 11
263 Skin barrier models (see Stratum corneum
Sebum lipid composition, 80 models)
Selenium in nails, 423 Skin barrier penetration (see Skin
Senile white hair, 324 penetration)
Separation of st. corneum lipids, 107 Skin diseases and st. corneum, 96
Single exposure to irritants, 219–222 Skin exposure and systemic effects, 17
Single gel phase model, 16, 22, 76, 128, Skin penetration and
137, 141, 154–162 atopic dermatitis, 197
Skin diseases, 197
dolphins, 173 drug delivery systems, lipid in, 27
feathers, 173 exposure, local and systemic effects,
fur, 173 17
hippopotamus, 173, 174 hydrophobic vs. hydrophilic pathways,
permeability, 186 16
quills, 173 ichthyosis, 197
scales, 173 moisturizers, 202
structure-function, 8 penetration enhancers, 16
thermoregulation, 186 percutaneous absorption, 199
whales, 173 psoriasis, 197
Skin barrier solvents, 202–206
amniotes, 179 stratum corneum, 197
amphibians, 176 transport mechanisms, 22
avians, 184 Skin structure epidermis, 5
evolution, 171 Skin structure stratum corneum, 5
fossorial adaptation, 182 Small-angle X-ray diffraction (SAXD)
frog, 176 lipid, 28
function of, 171 stratum corneum lipid, 28, 97
hippopotamus, 187 stratum corneum lipid, organization,
impact resistance, 173 101–103
invertebrates, 173 stratum corneum, structure, 109
lipid in stratum corneum, 11 Smoking and hair graying, 324
mammals, 182 Sodium lauryl sulfate (SLS), 220, 222,
naked mole rat, 182 225, 230–239
reptiles, 179 Solvents and
stratum corneum, 73 barrier properties, 204
structure, function, pp. 8–17 delipidization, 204
toxicology, 197 [Solvents and]
[Skin barrier] immediate skin reactions, 204
INDEX 449
immediate whitening, 204 water flux, 46

irritancy, 206 water and lipid phases, 46
irritant contact dermatitis, 204, 206 Stratum corneum function
nail rigidity, 422 atopic dermatitis, 117, 119
skin penetration, 202–206 desquamation, 159
stratum corneum lipid, 202–206 in diseases, 130
Spectroscopy and st. corneum lipids, 57 ichthyosis, 117, 119
Spingosides, 97 mechanical properties, 160
Stacked bilayers and st. corneum, 28 permeability, 175
Static secondary ion mass spectrometry proteins, 160
(SSIMS), 270 TEWL, 117
Stem cells, 9 waterproofing, 174
Steroid hormones (see Hair growth) Stratum corneum lipid mixtures
Sterols, 13 cholesterol, 123
Stratum basale, 9 cholesterol sulfate, 123
Stratum corneum phase behavior, 122
barrier function, 5 temperature, effect of, 122
barrier properties, 21 Stratum corneum lipid models, 108–109
chemical potential, 47 Stratum corneum lipid organization, 32,
corneocyte cell envelope, 131 43, 46, 68
corneocytes, 5 analysis of, techniques used, 128–129
corneodesmosomes, 5, 131 disorders in the lipid lattice, 103, 104
cosmetics, 9 electron diffraction, 104, 105, 113
desquamation, 96 lipid membranes, 14
diffusion pathways, 44 Lorenz factor, 103
epidermis, structure, 9 Miller indices, 106
formation of, 141–143, 174 SAXD, 101–103
impact resistance, 173 TEM, 104
intercellular lipids, 5, 7 (WAXD), 104
mechanical properties, 5 X-ray diffraction, 100–101
model membranes, 45, 46 Stratum corneum lipid phases
moisturizers, 9 electron diffraction, 115
molecular organization, 57 FTIR, 115
permeability, 199 WAXD, 115
pH gradient, 47 Stratum corneum lipids, 56
reconstructed epidermis, 96 acylceramide, 81
rhinos, 173 analytic techniques, 28
skin diseases, 96 AFM, 40, 42, 43
skin penetration, 197 barrier properties, 43
stress propagation, 173 bilayer configuration, 43
structure, 5, 5, 7, 20, 73 Brewster angle microscopy, 40
swelling, 5 ceramides, 13, 20, 32, 34, 75, 81, 97
temperature and lipid phases, 46 [Stratum corneum lipids]
[Stratum corneum] cholesterol, 20, 32, 34, 75, 82, 97, 98
transdermal drug delivery, 9, 21, 47, cholesterol esters, 13, 82
47, 96 cholesterol sulfate, 97
water, 8
450 INDEX
composition, confidence intervals, 73, solvents, 202–206

81–92 spectroscopy, 57
conformational changes, 148 stacked bilayers, 28
contamination, 80 sterols, 13
cornefied cell envelope, 82 TLC, 76–78
crystallization, lipid phases, 147 transport rate, 44
cubiclike membrane, 146 WAXD, 28, 97
diffusion barrier, 43 w-hydroxy acids, 75, 81
domain formation, 44 w-hydroxy ceramides, 14, 75, 81
epidermal cyst, 81 X-ray diffraction, 43, 56
extraction of, 116 Stratum corneum models
fatty acids, 13, 20, 32, 34, 75, 82, 97, barrier models, 57
98 barrier-forming process, pp. 0 –
fluorescence microscopy, 40 154
fluorescence quenching, 40 bricks and mortar, 14, 20
Fourier transform infrared cholesterol, 156
spectroscopy, 57, 65–68 conceptual models, 139
gas liquid chromatography (GLC), 79 differential scanning calorimetry, 69
GLC-electrospray ionization, 79 domain mosaic model, 5, 14, 22, 76
GLC-flame ionization detector, 79 energy needs, 140
GLC-mass spectrometry, 79 FTIR, 66, 67
grain boundaries, 130 in vivo gradients, 139
HPLC, 78 Landmann model, 141
HPTLC, 77 lipid composition, 139
6-hydroxysphingosine, 82 lipid phases, 68
identification of, 107 membrane folding model, 76, 137, 141,
lamellar bodies/granules, 97 143–146
light scattering detection, 78 model membranes, 68, 71
linoleic acid, 75 morphology, 140
lipid bilayers, 21 NMR, 59–65
lipid phase behavior, 22 sandwich model, 16, 100, 123–128
lipid phases, 28, 43 single gel phase model, 16, 22, 76, 128,
membrane permeability, 43 137, 141, 154–162
micro-Raman mapping, 43 water, 139
NMR, 28, 43, 57 Stratum corneum structure
permeability, 130 barrier, 96
phase diagrams, 40 corneocytes, 96
phase separation, 148 corneocytes, keratin, 113
phase tranformations, 130 cornified cell envelope, 75
physical properties, 22 diffraction curves, 110
pK, 43 diseases, 117
polar and nonpolar domains, 21 FFEM, 120
[Stratum corneum lipids] [Stratum corneum structure]
polymorphism, 129 FTIR, 113
rutthenium tetroxide staining, 110,117 in vivo, in vitro, 113
separation of, 107 intercellular lipid, 96
SAXD, 28, 97 keratin, 116
INDEX 451
lamellar bodies/granules, 75 Thermoregulation and skin, 186

lipid organization, 96, 99 Thin-layer chromatography (TLC)
lipid phase transistions, 115 iatroscan, 78
lipid phases, 113, pp. 109–117 quantitation, 77
SAXD, 109 stratum corneum lipid, 76–78
swelling, 113 Thioester bonds, 2
temperature, 110 Thyroid disorders, 359
thermodynamics, 137 Toxicology
TEM, 110 melanin, 308
water, 110 skin barrier, 197
WAXD, 110 Transdermal drug delivery, 9, 21, 27, 47,
Stratum granulosum, 9 47, 96
Stratum spinosum, 9 Transepidermal water loss (TEWL)
Stress and alopecia areata, 361 irritant contact dermatitis, 219
Stress propagation and st. corneum, 173 skin barrier function, 8, 11
Structure and function stratum corneum function, 117
hair follicle, 3, 251 Transmission electron microscopy (TEM)
nail, 375, 404 hair research, 263
skin, 8 lipids, 28
skin barrier, 8–17 nail structure, 411
stratum corneum, 20 stratum corneum lipid organization,
Structure of gray hair, 323 104
Sulfur content stratum corneum structure, 110
exocuticle, 274 Transport mechanisms and skin
hair keratin, 254 penetration, 22
keratinization, 1 Transport properties and domain mosaic
nail composition, 415, 419 model, 22
Superoxide dismutase, 235 Transport rate and st. corneum lipids, 44
Swelling of corneocytes, 5 Trichohyalin, 400
Swelling of st. corneum, 5, 113 Tumor necrosis factor- (TNF- )
alopecia areata, 364
Techniques, hair studies, 263 irritant contact dermatitis, 225
Techniques, nail studies, 0 Tyrosinase in melanosomes, 304
Telogen, 336
Telogen-anagen transition, 310 Ultrastructure and irritant contact
Temperature effects dermatitis, 219
lipid, aggregation, 27 Ultrastructure cuticle cell, 264
lipid organization, 99 UVR and hair color, 296
lipid phase transitions, 31
stratum corneum, 46 van der Waals and lipids, 99
[Temperature effects] VCAM-1, 232
stratum corneum lipids, 122 Ventral nail plate, 4
stratum corneum, structure, 110 Vertebrate skin barrier, 173
Testosterone metabolism, 350 Vitiligo
Thermodynamics and lipids, 137 alopecia areata, 359
Thermodynamic variables in domain hair pigmentary unit, disorders of, 326
formation, 40
452 INDEX
Water hydration force and lipids, 28 irritant contact dermatitis, 220

Water nail composition, 419
flux across stratum corneum, 46 particle probes, 220
and lipid phases in stratum corneum, skin, 220
46 X-ray photoelectron spectroscopy (XPS),
lipid phase transitions, 416 cuticle cell, 263, 270
nail composition, 416
in nail plate, 31
stratum corneum, 8
stratum corneum models, 139
stratum corneum, structure, 110
swelling and lipid aggregation, 24
Waterholding capacity and atopic
dermatitis, 200
Waterproofing function of st. corneum,
174
Weathering and fiber cuticle, 257
Whales
epidermis, 188
skin, 173
Wide-angle X-ray diffraction (WAXD)
keratin, 113
[Wide-angle X-ray diffraction
(WAXD)]
lipids, 28
stratum corneum lipid, 28, 97
stratum corneum lipid, organization,
104
stratum corneum lipid phases, 115
stratum corneum, structure, 110
Wool, references, 263
Xerosis in atopic dermatitis, 200

X-linked ichthyosis
nail composition, 423
X-ray diffraction
fatty acids, 39
lipid, 24
lipid monolyers, 39
lipid, aggregation, 22
nail structure, 405
nails, technique, pp. 405–411
stratum corneum lipid, 43, 56, 100–
101
X-ray diffraction and TEM,
nail structure, 412–415
X-ray microanalysis

Skin, Hair, and Nails, Forslind, 2005

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Skin, Hair, and Nails, Forslind, 2005

Uploaded by

Copyright:

Available Formats

Skin, Hair, and Nails

BASIC AND CLINICAL DERMATOLOGY

13. Immunomodulatory and Cytotoxic Agents in Dermatology, edited by Charles

MARCEL DEKKER, INC. NEW YORK • BASEL

ISBN 0-203-91286-1 Master e-book ISBN

ISBN: 0-8247-4313-X (Print Edition)

The integument—our first line of defense against a hostile physical environment

book should serve as an up-to-date reference for scientists in several disciplines

1. An Introduction to the Book 1

9. Skin Barrier Function in Diseased Skin and in Normal Skin 199

Part III Nails

Anders Boman, Ph.D. Occupational and Environmental Medicine, Stockholm

*Current affiliation: Chalmers University of Technology, Göteborg, Sweden.

Andrew G.Messenger, M.D., F.R.C.P. Department of Dermatology, Royal

*Current affiliation: Utrecht University, Utrecht, The Netherlands.

carrying a discontinuous medulla. In contrast, the eyelashes, being fibers that

The nail surface is smooth, sometimes even glossy. A scanning electron

Seldom are comparative aspects discussed in dermatological science.

FIGURE 1 Schematic presentation of a vertical section through epidermis.

FIGURE 2 A schematic drawing of the suggested epidermal proliferative unit. The

stratum corneum formerly was considered to be metabolic inactive, it hosts a

*Current affiliation: Utrecht University, Utrecht, The Netherlands.

FIGURE 3 Schematic illustration of the stratum corneum extracellular lipid bilayers,

[16, 17]. At very low concentrations, the amphiphiles exist as monomers in

FIGURE 5 Typical sequence of lipid morphologies obtained from lipid self-assembly in

incorporation of a simple inorganic salt such as NaSCN in the monoolein-water

The sequence in which the different structures appear can be described as a

FIGURE 6 Calorimetric temperature and sorption “scans” in correlation to the DMPC-

Ceramides are considered to be the most abundant lipid species in stratum

FIGURE 9 Hypothetical surface pressure-area ( s-A) isotherm including gaseous, liquid

The liquid expanded monolayer can be considered to be a fluid phase in which

conditions such as temperature, ionic strength, or osmotic pressure. This leads to

structure, cholesterol locates preferentially at the solid-liquid domain interfaces

model, specific transmembrane proteins may preferentially be located in the raft

presence of segregated domains of differing permeability can potentially explain

FIGURE 12 Schematic representation of the membrane consisting of stacked bilayers, in

FIGURE 13 Calculated water and solute flux through a DLPC-DMPC (XDMPC=0.5,

57. Lindblom, G., Johansson, L.B. and Arvidsson, G. Effect of cholesterol in

nature of lipid organization in a range of biomimetic systems. These studies are

FIGURE 1 Chemical structures of the SC model membrane components.

At equilibrium, there are more spins favoring the orientation parallel to H0

In a large static magnetic field, the quadrupolar interaction can be treated as a

Therefore, the energy differences between adjacent levels are

where “+” corresponds to m=0 1 transition, “–” corresponds to m=1 0

vQ=(3e2qQ/4) SCD, where SCD=<3 cos2 CD 1>/2, measuring the time-averaged

FIGURE 4 The 2H NMR spectrum as a function of temperature of the model SC

observed as membranes transform from one phase to another. In Figure 5, M1

identity of neighboring molecules in the model membrane. Hence in the three-

FIGURE 6 Infrared spectrum of SC model membrane containing ceramide, PA-d31, and

FIGURE 7 When a bilayer membrane undergoes a gel-liquid crystalline transition, a

FIGURE 8 The thermotropic behavior of the methylene stretching frequency of PA-d31

FIGURE 9 The lateral packing of isotopically alike lipid chains in an orthorhombic

“liquid ordered” phase, which is purported to be involved in the regulation of

Although some techniques applied to model membranes are bulk techniques,

4. Thewalt J, Kitson N, Araujo C, MacKay A, Bloom M. Models of stratum corneum

15. Mendelsohn R, Moore DJ. Vibrational spectroscopic studies of lipid domains in

A variation on conventional thin-layer chromatography is high-performance

substances. To compensate for this during quantitative work, researchers

FIGURE 1 Representative structures of the lipids of human stratum corneum.

TABLE 4 Change in Lipid Composition with Depth in Human Stratum Corneum

To our knowledge, the only studies to date that address, in a statistically

TABLE 5 Composition of Covalently Bound Lipids in Human Epidermal Stratum

*Covalently bound lipid fractions not included.