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Ceramides and Barrier Function in Healthy Skin

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Ceramides and Barrier Function in Healthy Skin Acta Derm Venereol 2010; 90: 350–353 INVESTIGATIVE REPORT Ceramides and Barrier Function in Healthy Skin Jakob Mutanu JUNGERSTED1, Lars I. HELLGREN2, Julie K. HØGH2, Tue DRACHMANN2, Gregor B.E. JEMEC1 and Tove AGNER3 1Department of Dermatology, University of Copenhagen, Roskilde Hospital, Roskilde, 2Department of System Biology and Centre for Advanced Food Sci- ence, Technical University of Denmark, Lyngby and 3Department of Dermatology, University of Copenhagen, Bispebjerg Hospital, Copenhagen, Denmark Lipids in the stratum corneum are key components in (9–13) has renewed interest in research into skin barrier the barrier function of the skin. Changes in lipid compo- function, including skin lipids. However, more infor- sition related to eczematous diseases are well known, but mation about the amount of SC lipids in normal skin is limited data are available on variations within healthy required in order to obtain a better understanding of the skin. The objective of the present study was to compare diseased skin. The aim of the present study was to eva- ceramide subgroups and ceramide/cholesterol ratios in luate the ceramide profile of healthy volunteers in rela- young, old, male and female healthy skin. A total of 55 tion to age and gender, and to correlate ceramide profile participants with healthy skin was included in the study. with TEWL and clinical perception of dry skin. Lipid profiles were correlated with transepidermal wa- ter loss and with information on dry skin from a ques- tionnaire including 16 people. No statistically significant MATERIALS AND METHODS differences were found between young and old skin for A total of 55 healthy volunteers was included in the study (19 ceramide subgroups or ceramide/cholesterol ratios, and men and 36 women). Thirty-two were < 40 years of age (median there was no statistically significant correlation between age 26 years, age range 18–39 years), 5 were between 40 and 60 years (median 51 years) and 18 were > 60 years of age (median answers about dry skin and ceramide levels. Interes- age 76 years, age range 61–88 years). Participants were enrolled tingly, a statistically significant higher ceramide/choles- after responding to posters at the local library and educational terol ratio was found for men than for women (p = 0.02). centre, and had no history of any major skin diseases. The study Key words: age; ceramides; ceramide/cholesterol ratio; was approved by the local ethics committee (SJ-7, 13986). gender; stratum corneum; TEWL. Methods (Accepted March 18, 2010.) SC was collected from all participants, using cyanoacrylate met- Acta Derm Venereol 2010; 90: 350–353. hods (14). Participants were instructed not to use any moisturi- zers on the day of the examination. The mid-volar forearm was Jakob Mutanu Jungersted, Department of Dermatology wiped with acetone to eliminate contamination with surface D92, Bispebjerg Hospital, Bispebjerg Bakke 23, DK- lipids. A drop of cyanoacrylate tissue-glue (LiquiBand®, Med- 2400 Copenhagen NV, Denmark. E-mail: Jungersted@ logic Global Ltd, Plymouth, UK) was placed on a glass slide and held tightly against the skin for 2 min, and then removed. gmail.com The slide was stored at –80°C until further analysis by high- performance thin layer chromatography (HPTLC). For HPTLC the skin lipids were separated on silica-coated HPTLC plates, The barrier function of the skin is located in the stratum due to the difference in the strength of interaction between the corneum (SC), where the majority of the very limited different lipids and the silica gel using a solvent mixture of transport of substances occurs between the corneocytes chloroform:methanol:acetic acid (190:9:1(v:v:v)). The samp- in the lipid bilayers (1, 2). The lipid bilayers are com- les were compared with standard curves made from ceramide posed of ceramides, cholesterol and free fatty acids, and 5 and cholesterol included on the plate. After separation, the plates were dried, stained with the fluorescent probe primuline the ceramide profile in particular has been related to the and the components were quantified through determination of barrier function of the skin. Ceramide 1 and ceramide 3 fluorescence intensity, as described in detail elsewhere (14). have been reported to be reduced in the SC of patients For ceramides we use the simple nomenclature (ceramide with atopic eczema (3–6), and some, but not all, studies 1–9); however, in Fig. 1 for clarity we have included the no- have reported a negative correlation between ceramide menclature suggested by Motta et al. (15). TEWL measurements were obtained at the volar forearm from 3 and transepidermal water loss (TEWL) (3, 7). Limited 31 of the volunteers. Measurements were performed on the op- data are available on variation in skin lipids and ceramide posite forearm to where the cyanoacrylate strip was taken, in profile due to physiological parameters such as age and accordance with guidelines (16). gender. Two studies have found overall reduced lipid Data on the clinical perception of dry skin was obtained content in aged human skin (4, 8), but research on healthy from 16 healthy participants (median age 32 years, age range 18–51 years; 5 males, 11 females). Individual dryness of the skin is very limited (2). skin, during the last week, expressed on a visual analogue scale Identification of filaggrin mutations related to atopic (VAS)-score (0 = no dry skin and 10 = severely dry skin), and eczema, ichthyosis and other eczematous diseases asked “Do you have dry skin (yes/no)? If yes, do you have dry Acta Derm Venereol 90 © 2010 The Authors. doi: 10.2340/00015555-0894 Journal Compilation © 2010 Acta Dermato-Venereologica. ISSN 0001-5555 Ceramides and healthy skin 351 % Age and ceramides 35 Age 18–40 (n = 32) Age > 60 (n = 18) 30 Fig. 1. Comparison of the percentage median 25 stratum corneum ceramide values for healthy young skin and healthy old skin. Percentiles 1 and 2 20 3 are shown for each median value. No statistically significant differences were measured. The y-axis 15 represents the median percentage distribution of ceramide 1–9 and is indicated left, while the 10 1 ceramide/cholesterol (Cer/Chol) ratio is indicated right. Motta’s nomenclature (15) is added for 5 each ceramide: A: α-hydroxy fatty acid; EO: ester-linked ω-hydroxyl acid; N: non-hydroxy 0 Cer 1 Cer 9+Cer 2 Cer 3 Cer 4 Cer 5+Cer 8 Cer 6 Cer 7 Cer/Chol fatty acid; P: phytosphingosine; S: sphingosine; EOS EOP+NS NP EOH AS+AH NH AP Ratio H: 6-hydrosphingosine. skin only in the winter or both summer and winter?” (Questions The questionnaire on dry skin showed no significant were asked and samples were obtained in October). differences in ceramide classes from volunteers with dry and normal skin, respectively, and no significant Statistics correlation between the VAS-score (0–10) and the dif- For comparison of differences between groups with respect to ferent ceramides was found. One in 5 males reported age and gender, the Mann-Whitney test was used. For correlation dry skin, compared with 7 of the 11 females. studies, the Spearman’s rank correlation coefficient was used. p-values < 0.05 were considered statistically significant. DISCUSSION RESULTS Ceramides are thought to play a major role in maintain- ing the efficient barrier function of the SC. Ceramide Results of the comparison of the ceramide profile in 1, in particular, is thought to be of importance in the young and old skin are illustrated in Fig. 1. No statisti- organization of lipids in the SC (1, 17, 18). However, cally significant differences in ceramides or ceramide/ knowledge of the different ceramides and ceramide/ cholesterol ratios (p = 0.57) were found in SC from cholesterol ratio in healthy skin is very limited, but is young (< 40 years) and old (> 60 years) participants. nevertheless important for a better understanding of Ceramide profiles from the SC of male and female diseased skin. volunteers are shown in Fig. 2. There was a significant No statistically significant difference was found in difference between the ceramide/cholesterol ratios for this study between young and old skin with respect to men and women (median men 2.0; median women 2.3; ceramide profile. Two other groups have previously p = 0.02). No statistically significant differences were studied ceramide changes with respect to age. One found between any of the ceramide subgroups between group examined Japanese volunteers and used roughly the age-matched men and women. the same cyanoacrylate method as ours. They found a The relationship with TEWL for ceramide 1 and 3 is decline in the total ceramide content with increasing age shown in Fig. 3. No significant correlation was found in SC, but did not differentiate among the subgroups of (p = 0.76 and p = 0.57, respectively). No correlation with ceramides (4), thus the results of this study are difficult TEWL was found for any of the other ceramide clas- to compare with our results, in which the ceramide ses either, and no correlation between TEWL and the levels are given as a ratio of cholesterol. The other ceramide/cholesterol ratio (p = 0.60) was found. group used tape-stripping on female Caucasians; they % Gender and ceramides 35 Fig. 2. Comparison of the percentage median Men (n = 19) Women (n = 36) 30 stratum corneum ceramide values for healthy 25 men and women and ceramide/cholesterol ratios (Cer/Chol). Percentiles 1 and 3 are shown for 2 20 each median value. No statistically significant 15 differences were found for the ceramide subgroups, but there was a statistically significant difference 10 1 in the Cer/Chol ratio between men and women. 5 The y-axis represents the median percentage 0 distribution of ceramide 1–7 and is indicated left, Cer 1 Cer 2 Cer 3 Cer 4 Cer 5 Cer 6 Cer 7 Cer/Chol while the Cer/Chol ratio is indicated right.
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