doi: 10.1111/j.1346-8138.2011.01406.x Journal of Dermatology 2012; 39: 295–300

ORIGINAL ARTICLE

Quantitative study of stratum corneum ceramides contents inpatients with sensitive skin

Hee Jin CHO, Bo Young CHUNG, Hee Bong LEE, Hye One KIM, Chun Wook PARK,

Cheol Heon LEE

Department of Dermatology, Collage of Medicine, Hallym University, Seoul, Korea

ABSTRACT

C

Y

R

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People with sensitive skin (SS) are those who state their skin is more sensitive than that of average persons. The stratum

corneum is responsible for maintaining skin barrier function. Ceramides, major constituents of stratum corneum lipids,

have been shown to predominantly contribute to the role. It has been suggested that barrier function in SS is decreased.

However, we could find very few reports about stratum corneum ceramides in SS. This study was done to find out differ-

ences in stratum corneum ceramides between SS and non-SS groups. Fifty individuals (20 with SS and 30 with non-SS)

were recruited. Lactic acid sting test (LAST) was performed on the left cheek. On six sites including the right cheek, arm,

thigh, leg, back and palm, transepidermal water loss (TEWL) and erythema index (EI) were measured. On the above six

sites, stratum corneum sheets were obtained by stripping with cyanoacrylate resin and stratum corneum lipids were

extracted, then, analyzed by high-performance liquid chromatography electrospray ionization mass spectrometry. LAST

scores were higher in the SS group, but not statistically significant. There were no differences in TEWL and EI values

between the two groups. The mean value of the quantity of stratum corneum ceramides on the face was significantly lower

in the SS group. On other sites, mean values were also lower in the SS group, but not statistically significant. The quantity

of ceramides was significantly decreased in the face of the SS group compared to that of the non-SS group. These results

suggest that the decrease in stratum corneum ceramides on facial skin could be related to SS development.

Key words: lactic acid sting test, sensitive skin, stratum corneum ceramides.

INTRODUCTION

Sensitive skin (SS) is a term with subjective and psychological conno-

tations, and is not clearly defined in dermatology. However, individu-

als with SS can react easily to external contactants, environmental

factors or internal stimuli, and develop exaggerated reactions or

dermatitis, compared to those with healthy skin. In general, SS is a

functional term used by individuals who show reduced tolerance

to frequent or prolonged use of cosmetics and toiletries with sensitive

reactions such as intolerable burning or itching sensation.1,2

Tests for diagnosing SS include subjective testing, which

involves determining the degree of skin irritation by applying stimu-

lants and evaluating the subject’s opinion, and objective testing,

which involves visual or mechanical observation. However, there is

no standardized method for diagnosing SS. The stratum corneum is

in direct contact with the external environment and acts as a skin

barrier, and the intercorneocyte lipids play important roles. Major

components of intercorneocyte lipids are ceramides, cholesterol

and fatty acids, and it has been reported that ceramides play the

essential role among them.3,4 Skin diseases can cause abnormali-

ties in the formation of intercorneocyte lipids. In particular, there

have been reports of decreased ceramides in the stratum corneum

orrespondence: Cheol H. Lee, M.D., Department of Dermatology,

oungdeungpo-gu, Seoul 150-950, Korea. Email: dermlee@yahoo.com, der

eceived 20 December 2010; accepted 25 July 2011.

2011 Japanese Dermatological Association

in skin diseases with a damaged skin barrier, such as atopic derma-

titis,5,6 psoriasis7 and ichthyosis.8 While the deterioration of skin

barrier function has been suggested in SS,1 there have been few

studies on ceramides in SS. This study was conducted to examine

the changes in the amount of ceramides in the stratum corneum

of SS.

METHODS

SubjectsA total of 50 individuals between 20 and 40 years of age were

selected and divided into SS and non-SS groups based on their

responses to the self-assessment questionnaire relating to SS.

Those being treated for diseases related to SS were excluded.

There were 20 subjects (six males and 14 females; average age,

29.5 years) in the SS group and 30 subjects (18 males and 12

females; average age, 30.5 years) in the non-SS control group.

MethodsWe measured the amount of ceramides in the stratum corneum,

and we also evaluated lactic acid sting test (LAST), transepidermal

water loss (TEWL) and erythema index (EI) in this study.

College of Medicine, Hallym University, 948-1 Daelim 1-dong,

mlee@yahoo.co.kr

295

Table 1. Analytical conditions of HPLC–MS for stratum corneumceramides

HPLC condition

Solvent A: 0.1% formic acidB: MeOH

Column Agilent Eclipse XDB-C18,

4.6 mm · 150 mm I.D.Column oven Ambient

Flow rate 0.6 mL ⁄ min

Injection volume 5 lL

Detection 220, 254 and 280 nmGradient (B %) 0 min (70%) – 3 min

(94%) – 9 min (97%) –

12 min (100%) – 15 min

(70%) – 25 min (70%)Mass spectrometer conditions

Sheath gas flow: 20

Aux sweep gas flow: 0I spray voltage (kV): 4.5

Capillary temp. (�C): 200�CCapillary voltage (V): )30.0

Tube lens: )10.0

HPLC, high-performance liquid chromatography; MS, mass spectrometry.

H.J. Cho et al.

LASTPrior to the test, the subjects washed their face and relaxed for

5–10 min. Lactic acid solution (10%, 50 lL) was lightly rubbed twice

on the left cheek (malar eminence) of the subjects using a cotton

swab, and the subjects were asked to rate the degree of stinging

they felt on a scale of 0–3 (0 = none, 1 = minor, 2 = moderate,

3 = severe) at 1-min intervals for a total duration of 10 min. Any

irritation rated 1 or greater during the 10-min interval was recorded

as a ‘‘stinger’’, and the average scores of the two groups were cal-

culated at each interval. The subjects washed their face with running

water after the test. The test was performed at room temperature of

19–24�C and relative humidity of 35–53%.

Measurement of TEWL and EIThe measurement of TEWL and EI was taken indoors after 30 min

of relaxation with the examination sites exposed. TEWL was

measured for 30–45 s with the probe of a Tewameter TM 210

(Courage + Khazaka, Koln, Germany),9 gently pressed on six

body surface sites (right cheek, forearm, thigh, leg, back and palm)

of the subjects, and EI was measured on the same sites by Derma-

spectrometer (Cortex Technology, Hadsund, Denmark)10 at room

temperature of 19–24�C and relative humidity of 35–53% at approx-

imately the same time in the afternoon.

Extraction of lipids in stratum corneum and analysis ofceramidesExtraction of lipids in stratum corneum. For collecting samples

of the stratum corneum, the above six sites in each subject were

gently swabbed with ethanol. One drop of cyanoacrylate resin (Aron

alpha; Toagosei, Tokyo, Japan) was placed on a glass slide, then

the glass slide was attached to the test site with slight pressure by

the same researcher, and detached carefully 1 min later. The

stripped stratum corneum sheet of 2.5 cm · 3.0 cm was attached

to a slide, raked with a blade and put into a small glass bottle. Hex-

ane : ethanol (95:5) mixed solution (5 mL) was added to the glass

bottle to melt the horny layer sheet, then 20 min of ultrasonication

(Sonicator; Sonic & Materials, Newtown, CT, USA) was conducted.

The content was subtracted with a syringe and membrane filtered

(MIllex, HV 0.45 lm; Millipore, St Charles, MO, USA) into a new

glass bottle. In filtrates, only the stratum corneum lipids are dis-

solved. The solvent was evaporated by nitrogen gas, then the

extract was kept at a temperature of )18�C.6

High-performance liquid chromatography mass spectrometry(HPLC–MS). The extract was dissolved adding 1 mL methanol,

moved to a 1.5-mL microtube and centrifuged to get the superna-

tant. The supernatant was filtered with the above membrane filtra-

tion and used as the sample for HPLC analysis.

The LCQ Advantage max system (Thermo Fisher, Waltham,

MA, USA) was used for HPLC analysis. The analysis column was

the Agilent Eclipse XDB-C18 (4.6 mm · 150 mm), with a flow rate

of 0.6 mL ⁄ min, and the volume of injected sample was 5 lL.

The mobile phase was analyzed with 0.1% formic acid (buffer A)

and methanol (buffer B) from 70%B to 100%B gradient for 25 min

and the detection was at ultraviolet 254 nm. For improvement of

mass ionization, 0.1% ammonium hydroxide (in water : metha-

296

nol = 1:1) was injected and analyzed at 3 lL ⁄ min velocity using a

Hitachi L-6200 pump (Merck-Hitachi, Darmstadt, Germany). The

mass spectrometer analytical conditions were: sheath gas flow,

20 arb; aux sweep gas flow, 0 arb; I spray voltage, 4.5 kV; capillary

temp, 200�C; capillary voltage, )30.0 V; and with positive ion mode

analysis (Table 1).11

Statistical analysisThe results were analyzed using SPSS ver. 14.0, the two groups

were compared with Student’s t-test, and the comparison of the

two groups in the LAST was conducted using the Mann–Whitney

U-test. The threshold of statistically significant difference was set

at P < 0.05.

RESULTS

LASTOf 50 subjects (20 in the SS group and 30 in the non-SS group), 16

in the SS group (80.0%) and 20 in the non-sensitive group (66.7%)

were classified as stingers because they felt a stinging sensation at

least once during the 10-min duration. Although the frequency of

stingers increased in the SS group, there was no statistical signifi-

cance (Table 2). As for the average scores of stinging sensation

recorded at 1-min intervals during 10 min, the SS group showed

higher scores than the non-sensitive group. Although there was no

statistical significance, the difference was increasing as time pro-

gressed and showed borderline significance in 10 min of stimulation

(P = 0.07) (Fig. 1).

TEWL and EIIn terms of TEWL measured at the six body surface sites, there was

no significant difference between the SS and non-SS groups

� 2011 Japanese Dermatological Association

Table 2. Positive rates of 10% lactic acid sting test between sensi-tive and non-sensitive skin groups

No. in SS (%) No. in NS (%) Total (%)

Stinger 16 (80) 20 (66.7) 36 (72)

Non-stinger 4 (20) 10 (33.3) 14 (28)

Total 20 30 50 (100)

Not statistically significant (P > 0.05). NS, non-sensitive skin group; SS,sensitive skin group.

Figure 1. Lactic acid sting test (LAST) scores over 10 min between

sensitive skin (SS) and non-SS (NS) groups. LAST scores in the SS

group were increasing and had borderline significance compared tothose in the NS group (P = 0.07).

Figure 2. Transepidermal water loss (TEWL) values between sensi-

tive skin (SS) and non-SS (NS) groups at six different sites. Therewere no significant differences in TEWL values between the groups

at each different body site.

Figure 3. Erythema index (EI) levels between sensitive skin (SS) andnon-SS (NS) groups at six different sites. There were no significant dif-

ferences in EI levels between the groups at each different body site.

Figure 4. (Top) High-performance liquid chromatogram (HPLC) of

one extracted stratum corneum lipid sample. The interval of retentiontime of 3.65–4.85 min was selected as that of ceramides. (Below) An

example of mass spectrometry–mass spectrometry pattern taken

from one peak in the HPLC chromatogram at a retention time of4.82 min.

Stratum corneum ceramides in sensitive skin

(Fig. 2). Moreover, the differences in TEWL values on the facial skin

between stingers and non-stingers with LAST in both sensitive and

non-SS groups were not statistically significant, and there was no

correlation between TEWL values and the results of LAST. EI values

measured at the six body surface sites neither showed any signifi-

cant difference between two groups (Fig. 3). The differences in EI

� 2011 Japanese Dermatological Association

values on the facial skin between stingers and non-stingers with

LAST in both sensitive and non-SS groups were not statistically

significant, and there was no correlation between EI values and

the results of LAST.

Comparison of ceramide quantity in the stratumcorneum lipidsWe performed HPLC–MS on the extracted stratum corneum

lipids. From the HPLC graph, we took the section between

297

Figure 5. Infrared spectrometry. The presence of ceramides was

proved by the N–H stretch of amide linkage near 3300 wavenumbers(per cm) in one extracted stratum corneum lipid sample.

Table 3. Comparison of the quantity of ceramides between sensitiveand non-sensitive skin groups at six different sites

Sites Group No. Values

Face* SS 20 3.70 · 108 ± 1.57 · 108

NS 30 4.95 · 108 ± 2.54 · 108

Forearm SS 20 3.65 · 108 ± 1.67 · 108

NS 30 4.26 · 108 ± 2.26 · 108

Thigh SS 20 3.80 · 108 ± 1.79 · 108

NS 30 4.67 · 108 ± 2.36 · 108

Leg SS 20 3.96 · 108 ± 1.58 · 108

NS 30 4.53 · 108 ± 2.58 · 108

Back SS 20 3.98 · 108 ± 1.97 · 108

NS 30 4.31 · 108 ± 1.93 · 108

Palm SS 20 4.16 · 108 ± 1.92 · 108

NS 30 4.62 · 108 ± 2.36 · 108

Quantity of stratum corneum ceramides in facial skin was significantlylower in the SS group compared to the NS group (*P < 0.05). Valuesrepresent mean ± standard deviation. NS, non-sensitive skin group; SS,sensitive skin group.

H.J. Cho et al.

the retention time of 3.65–4.85 min where the MS pattern of

ceramides was displayed, and the amount of ceramides was

calculated by measuring the area from the baseline to the peaks

(Fig. 4). In addition, at the infrared spectrometry that was con-

ducted separately, we were able to confirm ceramide materials

with N–H stretch frequency of amide linkage near 3300 wave-

numbers (per cm) (Fig. 5).

The mean value of the amount of stratum corneum cera-

mides on the facial skin of 20 subjects in the SS group was

3.70 · 108 ± 1.57 · 108, significantly lower than that of 30 sub-

jects in the non-SS group of 4.95 · 108 ± 2.54 · 108 (P = 0.037).

On other parts of body surface, such as the forearm, thigh, leg,

back and palm, the mean values of the amounts of stratum cor-

neum ceramides in the SS group were also lower than those in

the non-SS group but not statistically significant (P > 0.05)

(Table 3).

298

DISCUSSION

Many users of cosmetic products have experienced stinging and

itching sensation along with skin rash on their face. SS has been

defined by the cosmetic industry and consumers as a type of skin

that causes problems after using cosmetics. According to a Wes-

tern consumer marketing survey, approximately 40% of consumers

believed that they possessed the characteristics of SS. Whether a

person has SS depends solely on the patient’s comment. From the

perspective of dermatological science, however, SS refers to a type

of skin that is easily irritated by external preparations and prone to

irritation or allergic contact dermatitis compared to healthy skin.1,12

Fisher13 defined ‘‘status cosmeticus’’ as a state that makes any

cosmetics difficult to use due to sensitive reaction, and Amin and

Maibach14 introduced a concept of ‘‘cosmetic intolerance syn-

drome’’ for all cases of discomfort experienced using cosmetic

products. It is difficult to calculate exact statistics regarding SS

because its diagnosis has to depend entirely on the patient’s

account, but it has been reported that approximately 50% of

females and 30% of males claim that they have SS, indicating that

women are more sensitive to the symptoms.14–17

Sensitive skin can be defined in two ways. One is a subjective

term with only subjective symptoms such as stinging, burning or

pruritus without any skin lesions following various stimuli. Approxi-

mately 50% of patients with SS demonstrate these uncomfortable

symptoms without accompanying visible signs of inflammation. The

other is an objective term where subjective symptoms are accom-

panied by erythema, wheals, papules, vesicles, pustules and scales.

Skin lesions in these cases are often diagnosed as allergic con-

tact dermatitis, irritant dermatitis, seborrheic dermatitis or atopic

dermatitis.1,18.

Subjective judgment can be the effective differentiation for SS,

but does not provide accurate criteria. While various attempts have

been made to establish an objective means of substantiating patient

accounts, there have not been satisfying solutions. Subjective meth-

ods include LAST,19 the washing challenge test,20 and burning

induction test using chloroform ⁄ methanol solution.21 Objective

methods include measuring the EI or TEWL by applying sodium lau-

ryl sulfate,22 the erythema and wheal induction test using dimethyl-

sulfoxide23 and measuring the blistering time after applying

ammonium hydroxide on the skin.24 First introduced by Frosch and

Kligman,25 LAST is one of the most frequently used testing methods

for SS. Currently, the Hilltop chamber method proposed by Chris-

tensen and Kligman19 is widely used. In Korea, Ryu et al.16 used

LAST in their study on SS and Yang et al.26 in their research on ato-

pic dermatitis.

In order to eliminate confusion, Loffler et al.27 proposed that skin

susceptibility should be categorized into three types. It was sug-

gested that the skin irritancy based on subjective and objective defi-

nitions should be referred to as ‘‘sensistive skin’’ and ‘‘irritable

skin’’, respectively. They also regarded individuals that reacted

positively to chemical irritants (like lactic acid) as ‘‘stingers’’. They

claimed that there was no correlation between SS and irritable skin,

and that those with sensitive or irritable skin can be stingers. Our

study showed that the frequency of stingers was 80.0% (16 ⁄ 20 sub-

jects) in the SS group, which was higher than 66.7% (20 ⁄ 30 sub-

jects) in the non-SS group, but there was no statistical significance,

� 2011 Japanese Dermatological Association

Stratum corneum ceramides in sensitive skin

indicating that SS subjectively determined by patients has little cor-

relation with the degree of reaction to lactic acid stimulation. The

average degree of stinging sensation recorded at 1-min intervals for

10 min was higher in the SS group with no statistical significance.

However, the difference increased as time progressed and showed

borderline significance at 10 min of stimulation, which suggests that

further tests with a higher number of subjects and extended obser-

vation can yield significant results.

Although the pathophysiology of SS has yet to be clearly

explained, it has been reported that participating factors include

heightened neurosensory input signal, enhanced immune respon-

siveness and damaged skin barrier function.1,28 Examining TEWL

involves measuring the level of water loss from the epidermis, which

is a simple method of evaluating the damage of stratum corneum

barrier function. Pinnagoda et al.29 reported that SS patients

showed a greater level of TEWL than non-SS because the former

have a thinner and weaker stratum corneum than the latter. How-

ever, as with other studies conducted in Korea,17,30,31 this study did

not find out any differences in any test sites including the face

between the two groups. This indicates that while skin structural

characteristics like the thickness of the horny layer can be a factor,

we should also consider functional aspects such as different sen-

sory reactions to stimulation, release of inflammatory mediators and

the differences in their reactivity. Furthermore, we believe that TEWL

can be changed by moisture emanated through the sweat glands in

addition to the stratum corneum. The purpose of measuring the EI is

to examine the difference in sensory reaction to stimulation and

hyper-reaction of cutaneous blood vessels. In our study, the two

groups showed no difference in EI on various parts of the body

including the face. However, in a study conducted by Lee et al.31

where stimulators were swabbed on the forearm and observations

made at intervals, the EI of the SS group was significantly higher

than that of the non-SS group, demonstrating that SS is associated

with functional aspects, including excessive increase in the neuro-

sensory input signal, cutaneous vascular hyper-reactivity and vari-

ous inflammatory reactions.

Ceramides are a structurally heterogeneous and complex group

of sphingolipids containing derivatives of sphingosine bases in

amide linkage with a variety of fatty acids with 24–26 carbon

links.4,32 Inside a cell, it has been reported that ceramides act as

secondary signal transmitters and participate in the lipid signal

paths in mammal cells, and induce cell proliferation and differentia-

tion, restraint of cell cycle and apoptosis as major responses of

intra-cell reaction to external stimulation.33,34 Stratum corneum

ceramides are heterogeneous compound mixtures. Masukawa

et al.35 recently confirmed that more than 10 subtypes of approxi-

mately 340 ceramides exist as compound mixtures in human stra-

tum corneum lipids. The major components of the stratum corneum

lipids include ceramides, cholesterol and fatty acids, and it has been

reported that they combine to form a multi-lamellar structure, and

ceramides are major lipids that account for 40–50% of the stratum

corneum lipids and play essential roles in the structure and function

of the skin barrier.32,36

Non-invasive methods of obtaining the stratum corneum lipids

include in vivo surface extraction using organic solvents37 and tape

stripping.38 In our study, we used cyanoacrylate stripping,6 which

� 2011 Japanese Dermatological Association

involves applying cyanoacrylate resin on a glass slide and perform-

ing a single extraction of the horny layer, to obtain samples of the

stratum corneum lipids.

We analyzed the extracted stratum corneum lipids with HPLC

electrospray ionization mass spectrometry and MS. Having under-

gone MS, ceramides display MS patterns that can be categorized

as parent ceramide ion molecules and fragments of parent mole-

cules with small molecules detached from or attached to fragments

of sphingoid base, and fragments of fatty acids.39,40 We separately

performed infrared spectrometry, where certain groups of atoms

give rise to infrared bands at or near the same frequency regardless

of the structure of the rest of molecules, and the N–H stretch in

amides showed frequencies of approximately 3200–3400 wave-

numbers (per cm).41 From the infrared spectrometry conducted on

the extracted stratum corneum lipids, we were able to detect typical

N–H stretch frequencies of amide linkage near 3300 wavenumbers

(per cm), allowing us to confirm that the extracted stratum corneum

lipids contained ceramides. After performing HPLC–MS on the

extracted stratum corneum lipids, we took the section during the

retention time of 3.65–4.85 min on the graph where the MS pattern

of ceramides and molecular weights of 560–810 were displayed,

and the amount of ceramides was calculated by measuring the area

from the baseline. This study compared the average amounts of

ceramides in the stratum corneum on various parts of the body

(right cheek, forearm, thigh, leg, back, palm) between the SS group

and non-SS group. The results indicated that the mean values of

the amounts of ceramides in other parts of the body surface except

the face were lower in the SS group than the non-SS group, but the

difference was not statistically significant. However, on the face, the

SS group showed a statistically significant decrease in the mean

value of the amounts of ceramides compared to the non-SS, indi-

cating that the amount of ceramides in the stratum corneum on the

facial skin has a correlation with cases of SS on subjective judg-

ment. This result coincides with the findings of Christensen and Klig-

man16 and Ryu et al.19 that claimed subjective sensation is felt more

significantly in the facial area, especially around the cheeks, than

other parts of the body. Consequently, these results imply that

patients with SS have less ceramides in the stratum corneum

of facial skin than those with non-SS, and this can be associated

with the development of SS, suggesting the possibility that the

decreased facial stratum corneum ceramide is related to the

impaired barrier function of facial skin in patients with SS.

In conclusion, although SS is a common condition that causes

discomfort in 30–40% of the population, there is no clear dermato-

logical definition of the condition, and objective and standardized

diagnostic methods have yet to be established. While there have

been claims that the pathophysiology of SS is associated with

heightened neurosensory input, enhanced immune reaction and

damaged skin barrier function, none have been clearly substanti-

ated. In this study, the amount of ceramides, the major components

of the stratum corneum lipids, was significantly decreased in the

facial skin of the SS group compared to that of the non-SS group.

This result suggests that patients with SS have less ceramides in

the stratum corneum of facial skin than those with non-SS, and this

might be primarily associated with the development of SS, although

it has not been clearly identified yet and needs additional research.

299

H.J. Cho et al.

We believe that more organized studies on the skin barrier func-

tion of SS patients as well as the neurosensory mechanism of the

facial skin are necessary.

ACKNOWLEDGMENT

This study was supported by a grant of the Korean Health

Technology R&D Project, Ministry of Health & Welfare, Repub-

lic of Korea (No. A101550-1001-0000100).

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