Arch Dermatol Res (1991) 283 : 219 - 223

Archives of

9 Springer-Verlag 1991

Stratum corneum lipid abnormalities in atopic dermatitis A. Yamamoto, S. Serizawa, M. Ito, and Y. Sato Department of Dermatology, Niigata University School of Medicine, Niigata 951, Japan Received August 25, 1990

Summary. Patients with atopic dermatitis (AD) often present with a dry skin. To clarify the relationship between dry skin and lipid abnormalities within stratum corneum, stratum corneum lipids were collected from six AD patients aged 15 to 25 years and from sex- and age-matched controls. All major stratum corneum lipid classes were separated and quantitated by high-performance thin-layer chromatography/photodensitometry. Six ceramide fractions were also isolated and quantitated by thin-layer chromatography[photodensitometry. Esterified fatty acids of both ceramide 1 (acylceramides) and wax esters were analysed by capillary gas chromatography. The relative amounts of all the stratum corneum lipid classes including squalene, cholesterol esters, wax esters, triglycerides, free fatty acids, cholesterol, ceramides, cholesterol sulphate and phospholipids did not differ statistically between AD patients and controls. However, a significant decrease in proportion of ceramide 1, which is believed to be a carrier of linoleate responsible for a water-barrier function, and increased levels of esterified Cls: 1 fatty acids (oleate) of ceramide 1 were observed in AD patients. On the other hand, the fatty acid compositions as well as the proportions of C16:1 straight-chain component in sebum wax esters of AD patients were very similar to those of controls. These results suggest that a significantly reduced amount and/or structural alterations of ceramide 1 deriving from epidermal keratinocytes may be responsible for the impaired water-barrier function of the skin in AD. Key words: Ceramide 1 - Oleate - A t o p i c dermatitis Water-barrier function

late with the defect in the epidermal barrier [16, 22], a l t h o u g h the m e c h a n i s m o f the impaired water-barrier function in A D has n o t yet been clarified. Some reports [1, 16] have indicated that skin surface lipids in A D s h o w e d quantitative alterations, which m a y be responsible for the dry skin. In the present study, in order to investigate the pathogenesis o f dry skin in A D , the s t r a t u m c o r n e u m lipids o f A D patients were m e a s u r e d and c o m p a r e d with those o f c o n t r o l subjects. The subjects comprised pubertal children and y o u n g adults aged 15 to 25 years, whose sebum secretion rates are at high levels [30].

Materials and methods

Subjects The AD patients, diagnosed according to the criteria of Hanifin and Rajka [7], comprised three males and three females ranging from 15 to 25 years of age. All patients had clinically dry skin with no signs of eczema on their forearms except for the flexural region. The skin on the forearm had not been treated with any ointment afLer bathing 24 h previously. Six sex- and age-matched healthy subjects without clinical signs of AD or dry skin were similarly examined as controls.

Collection of stratum corneum lipids Stratum corneum lipids were collected from the forearms, except for the flexural region, by gentle rinsing with 250 ml of 95 % ethanol. The ethanolic extracts were concentrated in a rotary evaporator and then evaporated to dryness under a stream of nitrogen [1[5].

Separation and measurement of lipid classes Patients with atopic dermatitits ( A D ) often present with a dry skin [7]. The dry skin condition is very i m p o r t a n t in the aetiology o f A D since it m a y cause itching and resultant scratching [7]. This clinical s y m p t o m m a y corre-

Offprint requests to: A. Yamamoto

The lipid extracts were weighed, and determination of lipid classes was accomplished using high-performance thin-layer chromatography (HPTLC) separation [14]. Approximately 50 gg of each extract was applied to 10 x 20 cm HPLC plates (Merck, Darmstadt, Germany). The chromatogram was first developed to the 6.0-cm level with methanol/chloroform/water (20:95:1), and then to the 8.5-cm level with hexane/ether/acetic acid (80:20:10), and finally

220

A. Yamamoto et al. : Stratum corneum lipid abnormalities in atopic dermatitis %

10

Fig. 1. Typical charred thin-layer chromatogram of ceramides of a patient with atopic dermatitis. Ceramides migrate as a set of six distinct bands in the TLC system Table 1. Mean percentages (standard deviation) of stratum corneum lipids of patients with atopic dermatitis and control subjects Lipid fraction

Atopic dermatitis patients

Control subjects

Squalene Cholesterol esters Wax esters Triglycerides Free fatty acids Cholesterol Ceramides Cholesterol sulphate Phospholipids Others

23.0 3.0 12.2 10.3 8.3 14.0 15.0 3.6 4.8 5.8

21.3 (6.2) 3.1 (0.5) 11.0 (3.0) 9.1 (2.1) 8.0 (4.9) 14.9 (2.8) 16.8 (3.7) 3.2 (0.6) 5.0 (1.4) 7.6

(8.5) (0.4) (2.9) (1.0) (1.5) (2.9) (4.3) (0.3) (2.0)

Table 2. Mean percentages (standard deviation) of ceramides from the stratum corneum of patients with atopic dermatitis and control subjects Ceramides

Atopic dermatits patients

Control subjects

1 2 3 4 5 6

4.1 (2.8)* 6.0 (3.9) 23.3 (4.4) 10.9 (2.3) 27.3 (7.8) 27.8 (7.5)

9.9 12.2 20.5 8.9 26.3 21.9

(3.5) (4.3) (4.3) (7.9) (9.9) (4.0)

51! i 1:5

17

2'1

21

2>,

2~

F

M

F

F

M

M

Fig, 2. Ceramide 1 contents in stratum corneum lipids from the forearms of patients with AD and of sex- and age-matched controls. Shaded bars, AD patients; unshaded bars, control subjects. The numbers below the bars indicate the ages of the subjects. The paired t-test of mean values of the proportions showed a significant (p < 0.05) difference between the AD patients and control subjects. F, female; M, male

acetic acid (70: 30: I) [30]. The band corresponding to wax esters was detected by spraying the plate with 0.2% solution of 2',Tdichlorofiuorescein in ethanol and viewed under ultraviolet light. This band was scraped from the plate and the lipids were eluted with ether. The separation of cholesterol esters and wax esters was performed with this system, except for the third development. A band at the origin containing polar lipids was also scraped from the plate and the lipids were eluted with chloroform/methanol/water (50:50:1) [10, 15]. The polar lipid fraction was then analysed on a 0.25-mm-thick layer of silica gel G. The plate was developed twice with chloroform/methanol (19:1) [26], sprayed with 50% sulphuric acid, charred by heating at 150 ~C, and quantified by densitometry [30]. Figure 1 shows a typical charred thin-layer chromatogram of ceramides from an AD patient.

Preparation and analysis of f a t t y acid methyl esters

* p < 0.05 (paired t-test) with petroleum benzine to the top [12]. After drying, the chromatogram was sprayed with 10% (w/v) cupric sulphate hydrate in 8% (w/v) phosphoric acid and charred by heating at 180~ for 60rain [14]. After cooling, the charred chromatogram was quantitated by an absorbance reflection mode at 500 nm and zigzag scanning on a photodensitometer (CS-930, Shimadzu, Kyoto) equipped with an integrator (DR-2, Shimadzu). The amount of each lipid class was calculated by the densitometric area for cochromatographed lipid standards (Sigma, St. Louis, Mo., USA) [14].

Isolation o f ceramides and wax esters Approximately 50 mg of each extract was applied to a 20 x 20 cm thin-layer plate coated with a 0.5-ram-thick layer of silica gel G (Merck) that had been cleaned by development with chloroform/ methanol (2 : 1). The chromatogram was first developed with hexane, and then with benzene to the top of the plate. Relatively polar lipids on the plate were developed to the 10-cm level with hexane/ether/

Ceramide I was similarly separated from the polar lipid fractions by chromatography on a 0.5-mm-thick layer of silica gel G with a mobile phase. After detection with fluorescent indicator as described above, the band containing the ceramide 1 was scraped from the plate and lipids were eluted with chloroform/methanol (2:1)~ The isolated ceramide 1 was saponified with chloroform/methanol/10 N aqueous NaOH (2: 7:1) at 45~ for 1 h [15]. After acidifying with 2 N HC1, the liberated fatty acids were extracted into chloroform and isolated by preparative thin-layer chromatography (TLC) with a mobile phase of hexane/ether/acetic acid (70:30:1) [15] and then converted to fatty acid methyl esters (FAMEs) by heating at 80~ for 2 h in anhydrous 5% HC1 in methanol [30]. The FAMEs were purified by preparative TLC using toluene as developing solvent [15]. The dried fatty acid moiety of wax esters was converted to FAMEs under the same conditions as described above. For determination of the branched chain types in the wax esters, the FAMEs were separated by degrees ofunsaturation on argentated TLC plates, which had been dipped in aqueous 10% AgNO3, using toluene as developing solvent [18].

A. Yamamoto et al. : Stratum corneum lipid abnormalities in atopic dermatitis

221

Table 3. Mean percentages (standard deviation) of the esterified fatty acids of ceramide I and of sebaceous wax esters in patients with atopic dermatitis and control subjects Chain structures

Ceramide 1

Wax esters

Atopic dermatits patients

Control subjects

Atopic dermatitis patients

Control subjects

14:0 14:1 15:0 15:1 16:0 16:1 17:0 17:] 18:0 18:1 18:2 20:0

0.5 (0.4) 0.4 (0.3) 0.8 (0.6) 1.5 (0.4) 14.5 (5.2) 4.7 (2.8) 3.3 (0.6) 1.4 (1.4) 21.3 (6.4) 17.3 (7.5)* 28.2 (13.8) 5.9 (2.3)

1.5 0.5 1.3 1.7 17.6 3.8 2.6 6.6 19.7 10.7 29.7 3.9

(0.6) (0.2) (0.5) (1.5) (8.2) (2.0) (0.4) (3.2) (5.8) (5.0) (8.9) (1.2)

2.9 (1.6) 3.0 (1.6) 2.4 (0.5) 2.4 (0.6) 13.6 (4.6) 31.0 (10.1) 7.0 (0.1) 2.0 (1.8) 5.8 (2.3) 22.7 (11.4) 4.7 (3.9) n.d.

8.2(4.7) 2.5(1.9) 1.4(1.2) 3.3(1.1) 10.1 (2.6) 31.2 (7.9) 7.7(4.2) 0.9(1.6) 5.9(2.0) 22.7 (7.7) 4.3 (5.2) n.d.

Saturated Monounsaturated Diunsaturated

46.3 (14.4) 25.1 (11.3) 28.2 (13.8)

46.7 (12.6) 23.3 (8.3) 29.7 (8.9)

31.7 (3.7) 61.1 (6.5) 4.7 (3.9)

33.3 (4.8) 60.6(6.7) 4.3 (5.2)

* p < 0.05 (paired t-test) n.d., not detected

Gas chromatographic analysis of FAMEs The FAMEs prepared from both ceramide 1 and wax esters were analysed by gas chromatography using a 50 m fused silica HR-SS10 capillary column (Shinwakako, Kyoto, Japan), installed in a GC-9A (Shimadzu, Kyoto, Japan). The column temperature was programmed to start at 150~ and to increase at a rate of 3~ until a final temperature of 220~ had been attained [15]. Peaks were identified by relative retention times of authentic standards (Nu-Chek-Prep, Elysian, Minn., USA). Components with chain length less than 14 carbons or more than 20 carbons were not studied, as such components constituted only small proportions of the total [15, 30]. For determination of the branched chain types in the monounsaturated FAMEs, the prepared FAMEs were also chromatographed at 200~ on a 50 m fused silica OV-10t (Shimadzu) [3o].

Results The relative amounts of m a j o r stratum corneum lipids are shown in Table 1. N o significant differences between A D patients and control subjects were noted in all lipid fractions, but the proportions of ceramides were slightly lower in A D patients than in control subjects, while the opposite was true for purely sebaceous lipids (squalene, wax esters and triglycerides) [19]. The compositional data for the ceramides f r o m the stratum c o r n e u m of A D patients and control subjects are presented in Table 2. The proportions of both ceramide 1 and ceramide 2 were lower in A D patients than in control subjects. A statistically significant difference between the two groups was shown in the proportions of ceramide 1, but not for any other ceramide fraction. Figure 2 shows the proportion o f c e r a m i d e 1 for each A D patient c o m p a r e d with the sex- and age-matched controls. In five of the six comparisons, ceramide 1 f r o m the A D

patients showed much lower levels than those f r o m control subjects. Table 3 shows the compositions of the esterified fatty acids of ceramide i and of sebaceous wax esters. A m o n g the esterified fatty acids ranging from 14 to 20 carbons long, the Cls: 1 fatty acid of ceramide 1 was statistically higher in A D patients than in control subjects (Table 3, Fig. 3). The p r o p o r t i o n of C17: ~ fatty acid tended to be lower in A D patients than in control subjects; however, there was no significant difference between the two groups (Table 3). C o m p a r i n g the degree of saturation of ceramide 1, fatty acids in A D patients were slightly less saturated c o m p a r e d with those in control subjects. The constituents of the esterified fatty acids of the wax esters in A D patients and control subjects were very similar (Table 3). N o correlation was f o u n d between esterified fatty acids of ceramide I and those of wax esters in either A D patients or control subjects. Furthermore, the p r o p o r t i o n of Ca6: ~ straight-chain c o m p o n e n t as an index of sebaceous gland activity [30, 31] Was found to be identical in both groups (Table 4).

Discussion This study was designed to examine whether a relationship existed between the quality of lipids within the stratum corneum and dry skin in A D patients. Stratum corneum lipids, which form multiple b r o a d layers, are considered to be responsible for the water-barrier function [8, 9]. Although some abnormalities of specific stratum corneum lipids of epidermal origin, such as free cholesterol, cholesterol sulphate, shingolipids and linoleate, have been implicated in several scMy dermatose [3], it is u n k n o w n which lipid species regulate the barrier function.

222

A. Yamamoto et al. Stratum corneum lipid abnormalities in atopic dermatitis

A

C18:~ 0~8:2

B

c16:0

c18:0 ci8

CI8:1 016:o

18:1

C16:1

16:~

z4.L~...

%A/

I

I

I

I

I

I

I

I

I

I

0

5

10

15

20

0

5

10

15

20

Time (min)

Time (rain)

Fig. 3. Capillary gas chromatograms of the esterified fatty acid methyl esters, ranging from 14 to 20 carbons in length, of ceramide I fractions of a patient with AD (A) and a control subject (B). The peak for C18:1 fatty acid methyl esters is much higher in (A) than in (B) Table4. Mean percentages (standard deviation) of C1~=1 components in monounsaturated fatty acids from sebaceous wax esters of patients with atopic dermatitis and control subjects

CI6:1 straight C16:1 isobranched

Atopic dermatitis patients

Control subjects

39.2 (5.8) 10.6 (1.5)

39.6 (4.9) 10.5 (2.5)

In the present study, since the extracted stratum corneum lipids are of both epidermal and sebaceous origin, we were able to examine the relative amounts of all major lipid classes in stratum corneum [14]. As seen in Table 1, there was no significant difference in the lipid compositions between AD patients and controls; however, the proportion of ceramides was slightly lower in AD patients. Recently, a decreased ceramide fraction, expressed as a percentage of total lipids in stratum corneum, has been reported in the plantar skin of AD patients [13]. Moreover, the diminished water-barrier function after solvent treatment can be recovered by the application of ceramides [8]. These findings suggest that ceramides may have an important role in the barrier functions. Madison et al. [11] and Wertz et al. [25] have proposed a mechanism for the water-barrier function of the stratum corneum: ceramides derived from the glycolipids of membrane-coating granules (MCGs) may form lipid lamellar sheets which constitute the barrier [4]. The epidermal ceramides are composed of six different structural groups [27, 28]. Among them, ceramide 1 is considered to be responsible for the barrier function, because it is a carrier of linoleate and may serve uniquely as molecular rivets for the stabilization of the lipid lamellar sheets [2, 6, 25, 28]. The important role oflinoleate in the barrier function is evident in the study of dietary linoleate deficiency, in which MCGs fail to form lamellar sheets and waterbarrier function is diminished [5]. In this case, the

linoleate, which should be esterified to ceramide 1, is replaced by oleate [24], although this has not been proven in human epidermis. In the present study, it was found that ceramide 1 decreased proportionally with an increase in its esterified Ca8: a fatty acid in AD patients. Therefore, AD patients have altered ceramide constituents, so that the absolute amount of the linoleate is probably less than in normal controls. Presumably, the significant change in the proportion of the ceramide fraction may cause the structurally impaired lipid lamellar sheets in the stratum corneum, resulting in the diminished water-barrier function in AD patients. This hypothesis related to the mechanism of water-barrier dysfunction is strongly supported by an ultrastructural study which revealed the abnormalities of MCG in the transition zone between the stratum granulosum and stratum corneum in dry skin of AD patients [23]. In addition to the essential role of ceramides in waterbarrier function, we need to consider whether sebaceous lipids have any relationship with the affected barrierfunction in AD patients. Previous investigators have reported a decrease of sebum secretion rate in dry skin of AD patients [1, 16]. In contrast, in our study sebaceous lipids in AD patients tended to be higher than in control subjects (Table 1). To elucidate more clearly the sebum secretion rate in AD patients, we determined the fatty acid composition of wax esters, which are known to be the most suitable fraction for studying sebaceous fatty acids [7, 20, 2]]. We have previously shown that the proportions of C16:1 straight-chain components, which constitute a large proportion of the fatty acids of sebum wax esters, are positively correlated with sebum secretion rate [30]. Moreover, the proportions of C16:1 straightchain fatty acids showed a good correlation with urinary testosterone levels [31]. These results suggest that those sebaceous glands more actively producing lipid secrete sebum with a higher pro-

A. Yamamoto et al.: Stratum corneum lipid abnormalities in atopic dermatitis p o r t i o n o f C16 : 1 s t r a i g h t - c h a i n f a t t y a c i d in w a x esters. T h e r e f o r e , the p r o p o r t i o n o f C16:1 s t r a i g h t - c h a i n c o m p o n e n t in w a x esters is useful as a n i n d i c a t o r o f s e b a c e o u s g l a n d a c t i v i t y [31]. I n the p r e s e n t s t u d y , C16:1 straight-chain fatty acid showed almost identical prop o r t i o n s in b o t h A D p a t i e n t s a n d c o n t r o l subjects (Table 4). F u r t h e r m o r e , T a b l e 3 shows t h a t t h e r e is no significant difference in the p r o p o r t i o n o f m o n o u n s a t u r a t e d f a t t y acids in w a x esters b e t w e e n the two g r o u p s . T h e s e findings suggest t h a t A D p a t i e n t s m a y n o t h a v e a lower sebaceous gland activity than healthy people. T h e f a t t y acids o f s e b a c e o u s w a x esters c a n be i n c o r p o r a t e d i n t o the esterified f a t t y acids o f c e r a m i d e 1 [15, 19], a n d t o p i c a l l y a p p l i e d F A M E s c a n s i m i l a r l y affect the c o m p o s i t i o n s o f c e r a m i d e s [29]. This i n d i c a t e s t h a t the m o l e c u l a r s t r u c t u r e s o f c e r a m i d e s m a y be affected b y the c h a n g e o f s e b a c e o u s f a t t y acid c o m p o s i t i o n . F r o m o u r d a t a , however, t h e r e was n o evidence t h a t s e b a c e o u s f a t t y acids m i g h t affect the c o m p o s i t i o n o f the esterified f a t t y acids o f c e r a m i d e 1. T h e r e f o r e , t h e r e m a y be a p o s s i b i l i t y that AD patients have not only a disorder of ceramides b u t also a n a l t e r e d f a t t y a c i d m e t a b o l i s m in the e p i d e r m i s [12]. N e v e r t h e l e s s , since the f a t t y acid c o m p o s i t i o n o f o t h e r lipid classes, such as o t h e r c e r a m i d e f r a c t i o n s a n d free f a t t y acids, h a v e n o t b e e n e x a m i n e d , f u r t h e r studies are n e c e s s a r y to clarify the a b n o r m a l i t i e s o f f a t t y a c i d m e t a b o l i s m in the e p i d e r m i s o f A D p a t i e n t s .

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