Induction of a hardening phenomenon by repeated application of SLS: analysis of lipid changes in the stratum corneum.

Abstract

Adaptation of the skin to repeated influence of exogenous irritants is called the hardening phenomenon. We investigated the stratum corneum lipid composition before and after induction of a hardening phenomenon. Irritant contact dermatitis was induced in 23 non-atopic volunteers by repeated occlusive application of 0.5% sodium lauryl sulfate (SLS) over 3 weeks. At 3, 6 and 9 weeks after irritation, the SLS responses of pre-irritated skin and normal skin were compared. The horny layer lipid composition (ceramides 1-7, cholesterol and free fatty acids) was assessed before irritation and 3, 6 and 9 weeks after irritation. During the first 2 weeks of irritation the transepidermal water loss increased continuously and seemed to decrease during the third week (effect of adaptation). The barrier function of pre-irritated sites was more stable to SLS challenge. Three weeks after irritation, there was a significant increase of ceramide 1 (p<0.001). The only volunteer without hardening phenomenon showed no increase of ceramide 1. Ceramide 1 seems to play a key role as a protection mechanism against repeated irritation.

Full text

Page 1

INVESTIGATIVE REPORT
Induction of a Hardening Phenomenon by Repeated Application of
SLS: Analysis of Lipid Changes in the Stratum Corneum
Christian HEINEMANN1, Christiane PASCHOLD1, Joachim FLUHR1, Walter WIGGER-ALBERTI1,
Sibylle SCHLIEMANN-WILLERS1, Hany FARWANAH2, Klaus RAITH2, Reinhard NEUBERT2and Peter ELSNER1
1Department of Dermatology, Friedrich-Schiller-University Jena, Germany, and2Institute of Pharmaceutics and Biopharmaceutics, Martin-Luther-
University Halle, Germany
Adaptation of the skin to repeated influence of exogenous
irritants is called the hardening phenomenon. We
investigated the stratum corneum lipid composition before
and after induction of a hardening phenomenon. Irritant
contact dermatitis was induced in 23 non-atopic volunteers
by repeated occlusive application of 0.5% sodium lauryl
sulfate (SLS) over 3 weeks. At 3, 6 and 9 weeks after
irritation, the SLS responses of pre-irritated skin and
normal skin were compared. The horny layer lipid
composition (ceramides 1–7, cholesterol and free fatty
acids) was assessed before irritation and 3, 6 and 9 weeks
after irritation. During the first 2 weeks of irritation the
transepidermal water loss increased continuously and
seemed to decrease during the third week (effect of
adaptation). The barrier function of pre-irritated sites was
more stable to SLS challenge. Three weeks after
irritation, there was a significant increase of ceramide 1
(pv0.001). The only volunteer without hardening pheno-
menon showed no increase of ceramide 1. Ceramide 1
seems to play a key role as a protection mechanism
against repeated irritation. Key words: hardening
phenomenon; irritant contact dermatitis; stratum corneum
lipids; ceramides; transepidermal water loss.
(Accepted October 15, 2004.)
Acta Derm Venereol 2005; 85: 290–295.
Christian Heinemann MD, Department of Dermatology,
Friedrich Schiller University Jena, Erfurter Str. 35, DE-
07740 Jena, Germany. E-mail: christian.heinemann@
derma.uni-jena.de
Chronic irritant contact dermatitis (ICD), the most
frequent occupational disease of the skin, represents an
important socio-medical problem. Additionally, ICD is
known to be a risk factor for the development of the less
frequent allergic contact dermatitis (1).
Over the last decades, the epidemiology and patho-
genesis of ICD have been subject to intense investigation
(2, 3). It is well known that the hands are most
frequently concerned due to daily exposure to a wide
range of irritants at home or in the workplace (4).
However, only a few employees develop hand eczema,
while most employees, doing the same work and
exposed to the same irritants, do not. In the latter
group, transient dermatitis is often followed by a
long-lasting hyporeactivity to irritants. This is called
the hardening phenomenon and describes the adapta-
tion of the skin to repeated exogenous irritative noxes.
Widmer et al. (5) have induced hardening by repeated
irritation with sodium lauryl sulfate (SLS), but the
mechanisms of this phenomenon are still not fully
understood. However, for prevention of ICD and early
recognition of a predisposition, further knowledge about
the hardening phenomenon is of utmost importance.
The intact stratum corneum is the most important
structure for the skin barrier function. The bricks and
mortar model describes a two-compartment system of
lipid-depleted cells embedded in a uniquely organized
intercellular complex of multilamellar non-polar lipids
(6). During differentiation, keratinocytes synthesize
lipids which accumulate in lamellar granules. At the
interface of stratum granulosum and stratum corneum
the lipids are transported into the intercellular spaces.
After enzymatic processing, bilayers consisting of
ceramides, cholesterol and fatty acids are formed (7).
The lipid composition of the stratum corneum has been
described in detail (8–12).
There is some evidence that ceramides are particularly
important for the barrier function of the stratum
corneum (13). Exogenous application of ceramides led
to accelerated barrier regeneration (14). Imokawa et al.
(15) reported a decreased amount of ceramides in atopic
individuals. This study focused on the lipid composition
of the stratum corneum before and after induction of a
hardening phenomenon with special attention to the
different types of ceramides.
MATERIALS AND METHODS
Study population
The study was performed under standardized laboratory
conditions between December 2001 and May 2002.
Twenty-three healthy volunteers (11 women, 12 men; mean
age 26.7 (¡6.9) years; range 18–44 years) without any skin
disease participated in this study after obtaining a written
informed consent in accordance with the Helsinki declaration.
During the study period, the volunteers were allowed to
shower as usual but they had to avoid bathing, application of
detergents, moisturizers or emollients on their forearms and
Acta Derm Venereol 2005; 85: 290–295
Acta Derm Venereol 85
# 2005 Taylor & Francis. ISSN 0001-5555
DOI: 10.1080/00015550410026362

Page 2

upper arms. They were also asked to avoid sunbeds and solar
radiation. The study had passed review by the Ethical
Commission of the Friedrich Schiller University.
Procedures
Induction of cumulative irritation. Cumulative ICD was
induced at three sites (f2–f4) on the medial one-third of one
forearm and at three sites (u1–u3) on the medial side of one
upper arm by application of 230 ml of 0.5% aqueous solution
of SLS (Serva Feinbiochemie, Heidelberg, Germany) in
large Finn chambers (inner diameter 1.2 cm, Epitest Ltd,
Hyrla ¨, Finland) for 1 h on weekdays over a period of 3 weeks
(Fig. 1).
Challenge phase. Three, 6 and 9 weeks after the end of the
induction phase (on days 43, 64 and 85), an SLS irritation test
was performed. Each time a 1% aqueous solution of SLS was
applied for 24 h to one of the sites on the pre-treated upper
arm and to a previously untreated site on the opposite upper
arm (at the same distance from the elbow). Thus it was
possible to compare the reactivity of a pre-irritated skin site to
the reactivity of normal, previously non-irritated skin. The pre-
treated sites were identified for challenge by characteristic
anatomical features (such as veins and naevi), which were
marked for each volunteer on a separate plastic sheet for each
forearm and each upper arm.
Clinical evaluation and biophysical measurements. The mea-
surements were performed after the volunteers had rested for
at least 15 min and after acclimatization to the standardized
laboratory conditions (temperature 20–22˚C, humidity 20–
40%). They were made by the same observer at the same time
(¡1 h), 10 min before irritation. The following methods were
used. Visual scoring was performed according to Frosch &
Kligman (16). Transepidermal water loss (TEWL) as a valid
indicator of epidermal barrier function disruption by SLS
(17) was measured using the Tewameter TM 210H(Courage
& Khazaka, Cologne, Germany). The widely used TEWL
measurement using an open chamber system is based on
assessing the vapour pressure built up inside the probe tunnel
after application of the probe to the skin surface. The authors
are aware of possible errors of this method due to changes in
relative humidity and skin surface temperature (18). In order
to minimize errors, standard procedures were followed (19,
20). The intensity of erythema was quantified by using
the chromameter CR-300H(Minolta GmbH, Ahrensburg,
Germany) according to the recommendations of Elsner (21)
by exclusively considering the a*-value which represents the
red-green axis of the three-dimensional CIE L*-a*-b*-assay
system.
On day 1 of the induction phase all parameters were assessed
on fields f1–f4 and u1–u3. In the following the visual score was
assessed daily, whereas the TEWL and the chromametry a*
value were assessed on days 5, 12 and 19.
During the challenge phase (days 43, 64 and 85) all
parameters were assessed 10 min before and 10 min after
irritation with SLS on one of the fields u1–u3 and on the
corresponding field from u4–u6 (Fig. 1).
Extraction of the stratum corneum lipids. The lipid content
(ceramides 1–7, total ceramides, cholesterol, free fatty acids) of
the stratum corneum was assessed at four time points. The first
extraction was conducted on day 1 on the inner side of one
forearm (test side f1, not pre-irritated).
The other extractions took place 3, 6 and 9 weeks after
induction of the ICD on pre-irritated test sides (f2–f4). An
open glass ring with an inner diameter of 18 mm was pressed
to the skin on the test site and 6 ml of the extraction fluid
(acetone and diethylether 1:1) was pipetted into the lumen (15,
22). After 30 seconds the first fraction was removed (sample 1).
The procedure was repeated on the same test site and the
second fraction (sample 2) was removed 20 min after applica-
tion. The solution was mixed every 5 min using the pipette.
Superficial lipids (sebum lipids) were assumed to be washed off
with the first fraction, whereas the stratum corneum lipids
should be represented mainly in the second fraction. Both
fractions were stored for analysis in a glass tube at 220˚C.
The analysis was performed using automated multiple
development high performance thin-layer chromatography
(HPTLC) (23).
Chemicals for AMD-high performance thin-layer chromato-
graphy. Cholesterol, cholesteryl oleate and palmitic acid
(Sigma-Aldrich, Taufkirchen, Germany) and ceramide 3
(Cosmoferm, now Goldschmidt, Essen, Germany) were used
as reference substances to which the lipid quantifications were
related.
Solvents for TLC purposes were of analytical grade and
purchasedfromMerck(Darmstadt,
(Deventer, TheNetherlands)
Germany). Silica TLC plates (Kieselgel 60 F245, 20610 cm),
were supplied by Merck.
Germany),
Roth
Baker
and(Karlsruhe,
Application of the samples. The dried lipid extracts were first
dissolved in 500 ml of chloroform:methanol 1:1 (v/v); 50 ml of
each sample were applied automatically using the TLC
Sampler 4 (CAMAG, Muttenz, Switzerland). Band length,
first application position and distance from the bottom of the
plate were 8 mm, 16 mm and 8 mm, respectively.
Development of the plates and postchromatographic derivati-
zation. The lipid separation was carried out by means of an
AMD-2 apparatus (CAMAG, Muttenz, Switzerland) as
described previously (23). Briefly, an automated multiple
development procedure including 17 steps and based on
Fig. 1. Scheme of phase I (induction of irritant contact dermatitis (ICD)) and phase II (provocation and lipid extraction (L)). Test sites f1–f4:
forearms (f1 ceramide extraction on day 1, no irritation), test sites u1–u3: upper arm (phase I). Test sites f2–f4: forearms, preceding ICD; test sites
u1–u3: upper arm, preceding ICD; test sites u4–u6: opposite upper arm, without preceding ICD (phase II). V, visual score; T, trans-epidermal water
loss; C, chromametry, X, irritation using 0.5% SLS for 1 h and 1% SLS for 24 h (phase I and II, respectively). All measurements were assessed
before irritation.
Lipid organization of stratum corneum in irritant contact dermatitis
291
Acta Derm Venereol 85

Page 3

mixtures of chloroform, ethanol, acetone, n-hexane and
ethylacetate under acidic conditions was used to separate the
ceramide classes and the other lipids in the same run. After
drying, the plates were dipped into an aqueous solution of 10%
CuSO4, 8% H3PO4(v/v) and 5% methanol for 20 s and charred
in a drying oven at 150˚C for 30 min.
The visualized lipid bands were scanned using a TLC
Scanner 3 (CAMAG). The measurements were performed in
reflectance mode at a wavelength of 546 nm. The software
winCats was used to carry out the peak-based integration and
quantification. To avoid experimental errors, individual curves
of each reference lipid were set up for each HPTLC plate. As
standards are not commercially available for each ceramide
class, ceramide 3 was used as a standard for all ceramide
classes because of its average intensity as described previously
(24).
Statistics
Data were analysed using the statistical software program
SPSS 12.0 for Windows. For each test field, the mean values of
the chromametry and TEWL data points were calculated
from three and two measurements, respectively. The compa-
risons of the test and control data were performed with the
non-parametric Wilcoxon test. Alpha correction has been
conducted as measurements were made at three time points
and compared to the baseline values.
RESULTS
Induction of cumulative irritation
During the first 2 weeks of irritation a continuous
increase of TEWL, chromametry values and visual score
was observed (pv0.001). At the end of week 2, the
values reached a maximum (Fig. 2). The parameters on
the upper arm test sites reached higher values (especially
the TEWL), but generally showed a similar pattern
(data not shown).
Challenge phase
Three weeks after the induction phase the visual score
had returned to normal. There was no difference in the
TEWL between pre-irritated test sites and non-irritated
test sites before re-challenge at days 43, 64 and 85. After
re-challenge, the increase of TEWL was higher on non-
irritated test sites compared with pre-irritated sites at
day 65 (pv0.01; Fig. 3). The chromametry values
seemed to be higher on pre-irritated test sites at all time
points (Fig. 4).
Depending on the difference in TEWL response
between pre-irritated test sites and non-irritated test
sites the study population was divided into two groups:
those presenting a hardening phenomenon (negative
difference) and those without hardening phenomenon
(no difference or positive difference). At day 44, 16
volunteers were classified as having a hardening
phenomenon, 14 volunteers at day 65 and 15 volunteers
at day 86. Only one volunteer had no hardening
phenomenon at any time point, whereas eight volunteers
showed a hardening phenomenon in all challenges.
Lipid evaluation
Comparing the baseline stratum corneum lipid compo-
sition of volunteers presenting a hardening phenomenon
(n521) with the volunteer presenting no hardening
phenomenon (n51), ceramide 1 was lower in the
volunteers with hardening phenomenon than in the
volunteer without hardening phenomenon.
Lipids in sample 1 at days 44, 65 and 86
In 21 volunteers presenting a hardening phenomenon
the relative amount (as a percentage of the rest of the
stratum corneum lipids) of free fatty acids in the
extracted lipids was lower on challenge days (pv0.01
Fig. 2. Irritation phase on forearms, trans-epidermal water loss
(TEWL) values. Visual score, chromametry a*- and (g/m2/h) from
day 5 were compared to values from day 1, from day 12 to day 5 and
from day 19 to day 12 for statistically significant differences.
***pv0.001. Bars5SEM; n569.
Fig. 3. TEWL (g/m2/h) on pre-irritated test sites versus sites not pre-
irritated after challenge with 1% SLS occlusive for 24 h (phase II).
**pv0.01. Bars5SEM; n523.
292
C. Heinemann et al.
Acta Derm Venereol 85

Page 4

at day 86), whereas the relative amount of cholesterol
had increased (pv0.01 at day 65, pv0.001 at day 86)
(Table I). The total amount of lipids was significantly
higher at day 44 (pv0.001). In the single volunteer
without hardening phenomenon a loss of ceramide 1
and total ceramides was obvious on days 44 and 65.
Instead, there was an increased amount of free fatty
acids as compared with day 1 (data not shown).
Lipids in sample 2 at days 44, 65 and 86
In the 21 volunteers with hardening phenomenon the
total amount of lipids had significantly increased from
baseline to day 44 (pv0.01; data not shown).
The relative amount (as a percentage) of cholesterol
(pv0.01 at day 86) had increased, whereas the amount
of free fatty acids had decreased (pv0.05 at days 44 and
65 and pv0.01 at day 86). At day 44 there was a
significant increase of ceramide 1 (pv0.001; Table I). In
the single volunteer without hardening phenomenon
there was a decrease of ceramide 1 at days 44 and 65
(data not shown).
Analysis of the total amounts (in mg) of lipids showed
significantly increased
(pv0.001), c2 (pv0.05) and c4 (pv0.01) at day 44. c5
and c6 had increased at day 44 (pv0.05) and 65
(pv0.01), c7 at day 44 and 65 (pv0.01). The increase of
total ceramides was significant at day 44 (pv0.01) and
the increase of cholesterol at days 44 (pv0.001), 65
(pv0.05) and 86 (pv0.001). There were no significant
changes in the amounts of free fatty acids (Table I).
a amountof ceramide1
DISCUSSION
The concept of our study was based on a preceding
study by Widmer et al. (5). The question as to the origin
of the hyporeactivity of pre-irritated test sites remained
unsolved in their study. The authors discussed changes
in the lipid composition of the stratum corneum. To
further investigate the lipid changes after cumulative
irritation we additionally conducted lipid extraction
from pre-irritated test sites at different time points.
We were able to reproduce the increase of TEWL
during the first 2 weeks of the induction phase with a
slight decrease in the third week. Similar observations
were made by Gehring et al. (25) performing repetitive
washings on the forearms of non-atopic volunteers. At
day 65 we observed a significant difference between the
TEWL after re-challenge on pre-irritated sites and the
normal skin and evident difference at days 44 and 86.
This is also in accordance with the results of Widmer
et al. (5) and was interpreted as manifestation of a
hardening effect. Interestingly, the chromametry values
seemed to be higher on pre-irritated test sites after
re-challenge. This could be due to changes in the
responsiveness in the capillary system of the papillary
dermis after induction of irritation. McOsker & Beck
(26) hypothesized that a higher permeability of the
intercellular space and increased vessel activity might
lead to an accelerated wash out of irritating substances
in hardened skin.
Fig. 4. Chromametry delta a*-values on pre-irritated test sites versus
sites not pre-irritated after challenge with 1% SLS occlusive for 24 h
(phase II days 44, 65 and 86 – see Fig. 1). Bars5SEM; n523.
Table I. Volunteers (n521) with hardening phenomenon, samples 1 (relative amounts) and 2 (relative and total amounts); delta
values of the amounts of ceramides 1–7 (c1–c7), total ceramides, cholesterol and free fatty acids in % from day 1 to day 44, 65 and 86
Parameter
Sample 1 (% values)Sample 2 (% values)Sample 2 (mg values)
Day 44 Day 65Day 86Day 44 Day 65Day 86 Day 44Day 65 Day 86
c1
c2
c3
c4
c5
c6
c7
Total ceramides
Cholesterol
Free fatty acids
0.938
1.525
20.385
0.367
20.664
20.296
20.003
0.712
4.956
26.478
20.054
0.583
20.531
20.017
20.070
0.009
20.581
22.884
12.943**
28.434
20.033
0.069
20.734
20.069
20.949
20.579
20.234
24.146
11.024***
28.434**
2.384***
0.745
20.915
0.426
20.666
20.505
0.248
4.274
4.983
24.717*
0.964
1.168
20.015
0.365
0.444
0.328
1.666
3.748
5.194
210.673*
0.175
0.806
1.065
20.043
0.694
0.373
1.094
8.489
10.218**
29.026**
1.874***
1.395*
0.660
0.838*
1.525*
0.603*
1.521**
8.416**
7.271***
7.863
0.523
0.767
0.397
0.319
1.107**
0.43**
1.149**
4.692
3.647*
22.247
0.450
0.766
0.164
0.168
0.713
0.140
0.612
3.013
6.333***
23.478
*pv0.05, **pv0.01, ***pv0.001.
Lipid organization of stratum corneum in irritant contact dermatitis
293
Acta Derm Venereol 85

Page 5

The onset of hardening after the induction phase may
differ inter-individually as well as the duration of the
phenomenon (27). Therefore it was not unexpected that
some individuals showed a hardening phenomenon 3
weeks and some 6 weeks or 9 weeks after irritation.
Eight volunteers showed a hardening phenomenon at all
time points. In one volunteer we determined a hardening
phenomenon after 3 weeks, none at 6 weeks and again at
9 weeks using our definition. We suppose that in this
individual the challenge at 6 weeks on the normal skin
was not conducted properly, as the TEWL increased to
85 and 106 g/m2/h on normal skin at day 44 and day 86
but only to 49 g/m2/h at day 65.
There are few data about the prevalence of hardening
phenomenon but an investigation in hairdressing
apprentices showed that 90% of those suffering from
alkali-eczema at the beginning, cleared during training
period. Only 10% finished by presenting the typical
hairdressers’ hand eczema (28, 29). Schwanitz (30)
deducted a prevalence of 90% of hardening phenom-
enon within this population. Not all individuals are able
to adapt to repeated irritation, atopic patients seem to
develop hyporeactivity to a lower degree than non-
atopics (25, 31). Therefore it is not surprising that only
one volunteer in our study population did not show a
hardening phenomenon at any time point (atopic
individuals were excluded by the study protocol).
Onset of hardening later than 9 weeks after irritation
seems unlikely, so we compared the lipid profile of this
one volunteer without hardening phenomenon to the
average of the remaining study population. In the
samples at day 1, the baseline ceramide 1 was lower in
volunteers presenting a hardening phenomenon. This
suggests that it is not possible to predict ability to develop
hyporeactivity by measuring baseline ceramide 1. After
induction of irritation the production of ceramide 1
was enhanced in volunteers with hardening pheno-
menon. On the contrary, in the individual without
hardening effect, the amount of ceramide 1 had
decreased over the time. However, more volunteers
without hardening phenomenon would be necessary to
deduct conclusions from the comparison of the stratum
corneum lipid composition between the two groups.
The stratum corneum lipids were collected in two
fractions. In sample 1, which should contain the major
part of the superficial lipids (including sebum lipids), the
relative amount of total ceramides and free fatty acids in
the extracted lipids was lower on days 44, 65 and 86
compared with the baseline, whereas the relative amount
of cholesterol had increased. The role of surface lipids
for the epidermal permeability barrier function is still
not well defined (22) and was not subject to investiga-
tion in this study. Sample 2 should reflect more properly
the stratum corneum lipids, as the contaminating
surface lipids were removed with sample 1. Compared
with the baseline, the relative amount of total ceramides
(not significant) and cholesterol in this fraction had
increased and the free fatty acids were lower at all
challenge days. The total amount of lipids was increased
significantly at day 44. This is in accordance with the
findings of Proksch et al. (32, 33), who observed an
increase in skin lipid synthesis after irritation. They
observed an augmented synthesis of cholesterol after
acute irritation and an increase in ceramides after
chronic irritation. Interestingly, the relative and total
amount of ceramide 1 had increased significantly at
day 44 and seemed to decline over the following
study period. Ceramide 1 is the least polar of the
stratum corneum ceramides and probably plays a
major role in determining the state of organization
of the intercellular bilayers (34, 35). It is responsible
for the formation of lipid bilayers and their stability (7).
Di Nardo et al. (13) investigated the relationship
between the ceramide content of the stratum corneum
and the reactivity to SLS. In their findings the basic
content of ceramide 1 and the TEWL 24 h after
irritation with 3% SLS was correlated negatively in a
linearfunction.Other authors
amounts of ceramide 1 in atopic individuals and
patients suffering from lamellar ichthyosis (15, 36–38).
Our findings suggest that there is an upregulation of
the production of ceramide 1 in response to chronic
irritation. Upregulation of ceramide 1 synthesis seems
to play a major role in the development of a
hardening effect.
reported reduced
REFERENCES
1. Smith HR, Basketter DA, McFadden JP. Irritant derma-
titis, irritancy and its role in allergic contact dermatitis.
Clin Exp Dermatol 2002; 27: 138–146.
2. Diepgen TL, Coenraads PJ. The epidemiology of occupa-
tional contact dermatitis. Int Arch Occup Environ Health
1999; 72: 496–506.
3. Denig NI, Hoke AW, Maibach HI. Irritant contact
dermatitis. Clues to causes, clinical characteristics, and
control. Postgrad Med 1998; 103: 199–200, 207–208,
212–213.
4. Nettis E, Colanardi MC, Soccio AL, Ferrannini A,
Tursi A. Occupational irritant and allergic contact
dermatitisamong healthcare
Dermatitis 2002; 46: 101–107.
5. Widmer J, Elsner P, Burg G. Skin irritant reactivity
following experimental cumulative irritant contact derma-
titis. Contact Dermatitis 1994; 30: 35–39.
6. Menon GK, Brown BE, Elias PM. Avian epidermal
differentiation: role of lipids in permeability barrier
formation. Tissue Cell 1986; 18: 71–82.
7. Wertz PW. Lipids and barrier function of the skin. Acta
Derm Venereol Suppl 2000; 208: 7–11.
8. Madison KC. Barrier function of the skin: ‘‘la raison
d’etre’’ of the epidermis. J Invest Dermatol 2003; 121:
231–241.
9. Wertz PW, Downing DT. Ceramides of pig epidermis:
structure determination. J Lipid Res 1983; 24: 759–765.
workers. Contact
294
C. Heinemann et al.
Acta Derm Venereol 85

Page 6

10. Wertz
Downing DT. The composition of the ceramides from
human stratum corneum and from comedones. J Invest
Dermatol 1985; 84: 410–412.
11. Bouwstra J, Pilgram G, Gooris G, Koerten H, Ponec M.
New aspects of the skin barrier organization. Skin
Pharmacol Appl Skin Physiol 2001; 14(Suppl 1): 52–62.
12. Bouwstra JA, Dubbelaar FE, Gooris GS, Weerheim AM,
Ponec M. The role of ceramide composition in the lipid
organisation of the skin barrier. Biochim Biophys Acta
1999; 1419: 127–136.
13. di Nardo A, Sugino K, Wertz P, Ademola J, Maibach HI.
Sodium lauryl sulfate (SLS) induced irritant contact
dermatitis: a correlation study between ceramides and in
vivo parameters of irritation. Contact Dermatitis 1996; 35:
86–91.
14. Zettersten EM, Ghadially R, Feingold KR, Crumrine D,
Elias PM. Optimal ratios of topical stratum corneum
lipids improve barrier recovery in chronologically aged
skin. J Am Acad Dermatol 1997; 37: 403–408.
15. 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.
16. Frosch PJ, Kligman AM. The soap chamber test. A new
method for assessing the irritancy of soaps. J Am Acad
Dermatol 1979; 1: 35–41.
17. Fluhr JW, Kuss O, Diepgen T, Lazzerini S, Pelosi A,
Gloor M, et al. Testing for irritation with a multifactorial
approach: comparison of eight non-invasive measuring
techniques on five different irritation types. Br J Dermatol
2001; 145: 696–703.
18. Norlen L, Engblom J, Andersson M, Forslind B. A new
computer-based evaporimeter system for rapid and precise
measurements of water diffusion through stratum cor-
neum in vitro. J Invest Dermatol 1999; 113: 533–540.
19. Rogiers V. EEMCO guidance for the assessment of
transepidermal water loss in cosmetic sciences. Skin
Pharmacol Appl Skin Physiol 2001; 14: 117–128.
20. Pinnagoda J, Tupker RA, 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.
21. Elsner P. Chromametrie. Hardware, measuring principles
and standardization of measurements. In: Berardesca E,
Elsner P, Maibach HI, eds. Handbook of skin bio-
engineering. Cutaneous blood flow and erythema. Boca
Raton: CRC Press, 1994.
22. Imokawa G, Akasaki S, Hattori M, Yoshizuka N.
Selective recovery of deranged water-holding properties
by stratum corneum lipids. J Invest Dermatol 1986; 87:
758–761.
PW, MiethkeMC, Long SA,StraussJS,
23. Farwanah H, Neubert R, Zellmer S, Raith K. An
improved procedure for the separation of major stratum
corneum lipids by means of automated multiple develop-
ment thin-layer chromatography. J Chromatogr B Analyt
Technol Biomed Life Sci 2002; 780: 443–450.
24. Weerheim AM, 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.
25. Gehring W, Gloor M, Kleesz P. Predictive washing test for
evaluation of individual eczema risk. Contact Dermatitis
1998; 39: 8–13.
26. McOsker DE, Beck LW. Characteristics of accommodated
(hardened) skin. J Invest Dermatol 1967; 48: 372–383.
27. Wulfhorst B. Hardening-Effekt. Teil I. U¨berblick und
Stand der Forschung. Allergologie 1996; 11: 500–508.
28. Borelli S, Manok M. Ergebnisse von Untersuchungen bei
Berfusanfa ¨ngern im Friseurgewerbe. Berufsdermatosen
1961; 9: 271–276.
29. Borelli S, Moormann J, Du ¨ngemann H, Manok M.
Ergebnisse einer vierja ¨hringen Untersuchungsreihe bei
Berufsanfa ¨ngern des Friseurgewerbes. Berufsdermatosen
1965; 13: 213–239.
30. Schwanitz HJ. Das atopische Palmoplantarekzem. Berlin:
Springer-Verlag, 1986.
31. Wulfhorst B. Hardening-Effekt. Teil II. Experimentelle
InduktiondurchIrritantien.
541–549.
32. Proksch E, Holleran WM, Menon GK, Elias PM,
Feingold KR.Barrierfunction
lipid and DNA synthesis. Br J Dermatol 1993; 128:
473–482.
33. Proksch E. [Regulation of the epidermal permeability
barrier by lipids and hyperproliferation]. Hautarzt 1992;
43: 331–338 [in German].
34. Bouwstra JA, Gooris GS, Dubbelaar FE, 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.
35. Kuempel D, Swartzendruber DC, Squier CA, Wertz PW.
In vitro reconstitution of stratum corneum lipid lamellae.
Biochim Biophys Acta 1998; 1372: 135–140.
36. Matsumoto M, Umemoto N, Sugiura H, Uehara M.
Difference in ceramide composition between ‘‘dry’’ and
‘‘normal’’ skin in patients with atopic dermatitis. Acta
Derm Venereol 1999; 79: 246–247.
37. Di Nardo A, Wertz P, Giannetti A, Seidenari S. Ceramide
and cholesterol composition of the skin of patients with
atopic dermatitis. Acta Derm Venereol 1998; 78: 27–30.
38. Yamamoto A, Serizawa S, Ito M, Sato Y. Stratum
corneum lipid abnormalities in atopic dermatitis. Arch
Dermatol Res 1991; 283: 219–223.
Allergologie1996; 12:
regulates epidermal
Lipid organization of stratum corneum in irritant contact dermatitis
295
Acta Derm Venereol 85