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Keywords:

  • lactic acid sting test;
  • sensitive skin;
  • stratum corneum ceramides

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

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 differences 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.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

Sensitive skin (SS) is a term with subjective and psychological connotations, and is not clearly defined in dermatology. However, individuals 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 stimulants 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 abnormalities in the formation of intercorneocyte lipids. In particular, there have been reports of decreased ceramides in the stratum corneum in skin diseases with a damaged skin barrier, such as atopic dermatitis,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

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

Subjects

A 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.

Methods

We 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.

LAST

Prior to the test, the subjects washed their face and relaxed for 5–10 min. Lactic acid solution (10%, 50 μL) 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 calculated 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 EI

The 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, Köln, 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 Dermaspectrometer (Cortex Technology, Hadsund, Denmark)10 at room temperature of 19–24°C and relative humidity of 35–53% at approximately the same time in the afternoon.

Extraction of lipids in stratum corneum and analysis of ceramides

Extraction 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. Hexane : 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 μm; Millipore, St Charles, MO, USA) into a new glass bottle. In filtrates, only the stratum corneum lipids are dissolved. 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 supernatant. The supernatant was filtered with the above membrane filtration 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 μL. 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 : methanol = 1:1) was injected and analyzed at 3 μL/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

Table 1.   Analytical conditions of HPLC–MS for stratum corneum ceramides
  1. HPLC, high-performance liquid chromatography; MS, mass spectrometry.

HPLC condition
 SolventA: 0.1% formic acid
B: MeOH
 ColumnAgilent Eclipse XDB-C18, 4.6 mm × 150 mm I.D.
 Column ovenAmbient
 Flow rate0.6 mL/min
 Injection volume5 μL
 Detection220, 254 and 280 nm
 Gradient (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: 0 I spray voltage (kV): 4.5 Capillary temp. (°C): 200°C Capillary voltage (V): −30.0 Tube lens: −10.0

Statistical analysis

The 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

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

LAST

Of 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 significance (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 progressed and showed borderline significance in 10 min of stimulation (= 0.07) (Fig. 1).

Table 2.   Positive rates of 10% lactic acid sting test between sensitive and non-sensitive skin groups
 No. in SS (%)No. in NS (%)Total (%)
  1. Not statistically significant (> 0.05). NS, non-sensitive skin group; SS, sensitive skin group.

Stinger16 (80)20 (66.7)36 (72)
Non-stinger4 (20)10 (33.3)14 (28)
Total203050 (100)
image

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 to those in the NS group (= 0.07).

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TEWL and EI

In terms of TEWL measured at the six body surface sites, there was no significant difference between the SS and non-SS groups (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 significant difference between two groups (Fig. 3). The differences in EI 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.

image

Figure 2.  Transepidermal water loss (TEWL) values between sensitive skin (SS) and non-SS (NS) groups at six different sites. There were no significant differences in TEWL values between the groups at each different body site.

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image

Figure 3.  Erythema index (EI) levels between sensitive skin (SS) and non-SS (NS) groups at six different sites. There were no significant differences in EI levels between the groups at each different body site.

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Comparison of ceramide quantity in the stratum corneum lipids

We performed HPLC–MS on the extracted stratum corneum lipids. From the HPLC graph, we took the section between 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 conducted separately, we were able to confirm ceramide materials with N–H stretch frequency of amide linkage near 3300 wavenumbers (per cm) (Fig. 5).

image

Figure 4.  (Top) High-performance liquid chromatogram (HPLC) of one extracted stratum corneum lipid sample. The interval of retention time 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 of 4.82 min.

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image

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.

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The mean value of the amount of stratum corneum ceramides on the facial skin of 20 subjects in the SS group was 3.70 × 108 ± 1.57 × 108, significantly lower than that of 30 subjects in the non-SS group of 4.95 × 108 ± 2.54 × 108 (= 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 corneum ceramides in the SS group were also lower than those in the non-SS group but not statistically significant (> 0.05) (Table 3).

Table 3.   Comparison of the quantity of ceramides between sensitive and non-sensitive skin groups at six different sites
SitesGroupNo.Values
  1. Quantity of stratum corneum ceramides in facial skin was significantly lower in the SS group compared to the NS group (*< 0.05). Values represent mean ± standard deviation. NS, non-sensitive skin group; SS, sensitive skin group.

Face*SS203.70 × 108 ± 1.57 × 108
NS304.95 × 108 ± 2.54 × 108
ForearmSS203.65 × 108 ± 1.67 × 108
NS304.26 × 108 ± 2.26 × 108
ThighSS203.80 × 108 ± 1.79 × 108
NS304.67 × 108 ± 2.36 × 108
LegSS203.96 × 108 ± 1.58 × 108
NS304.53 × 108 ± 2.58 × 108
BackSS203.98 × 108 ± 1.97 × 108
NS304.31 × 108 ± 1.93 × 108
PalmSS204.16 × 108 ± 1.92 × 108
NS304.62 × 108 ± 2.36 × 108

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

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 Western 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 syndrome” 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. Approximately 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 accompanied by erythema, wheals, papules, vesicles, pustules and scales. Skin lesions in these cases are often diagnosed as allergic contact 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 methods 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 lauryl sulfate,22 the erythema and wheal induction test using dimethylsulfoxide23 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 Christensen 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 atopic dermatitis.

In order to eliminate confusion, Löffler et al.27 proposed that skin susceptibility should be categorized into three types. It was suggested that the skin irritancy based on subjective and objective definitions 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 subjects) in the SS group, which was higher than 66.7% (20/30 subjects) in the non-SS group, but there was no statistical significance, indicating that SS subjectively determined by patients has little correlation 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 observation 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 responsiveness 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. However, 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 sensory 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 neurosensory input signal, cutaneous vascular hyper-reactivity and various 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 differentiation, 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 approximately 340 ceramides exist as compound mixtures in human stratum 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 involves applying cyanoacrylate resin on a glass slide and performing 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 undergone MS, ceramides display MS patterns that can be categorized as parent ceramide ion molecules and fragments of parent molecules 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 wavenumbers (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, indicating that the amount of ceramides in the stratum corneum on the facial skin has a correlation with cases of SS on subjective judgment. This result coincides with the findings of Christensen and Kligman16 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 dermatological 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 substantiated. 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.

We believe that more organized studies on the skin barrier function of SS patients as well as the neurosensory mechanism of the facial skin are necessary.

ACKNOWLEDGMENT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES

This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (No. A101550-1001-0000100).

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENT
  8. REFERENCES
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