Introduction

The outermost layer of the skin, the stratum corneum, consists of corneocytes embedded in a complex matrix of multilamellar organized lipids containing mainly ceramides, cholesterol and free fatty acids [10, 11, 16].

The corneocytes have a peripheral rigid protein envelope, the so-called cornified envelope [25], to which a lipid monolayer (the cornified lipid envelope) is covalently attached [45]. While the intercellular lipids can be easily extracted by organic solvents, the covalently bound lipids need first to be hydrolyzed under alkaline conditions [45].

Ceramides, which comprise the major constituents of the free extractable stratum corneum lipids, are known to play a key role in structuring and maintaining the epidermal barrier function of human skin [7, 20]. They consist of sphingoid bases which are amide-linked to α hydroxylated, ω hydroxylated or non-hydroxylated fatty acids. Separation on high performance thin layer chromatography (HPTLC) plates shows seven ceramide classes. According to a nomenclature proposed previously [30, 38], they were recognized as Cer [EOS], [NS], [NP], [EOH], [AS], [AP], and Cer [AH]. The ceramide [AS] fraction, however, was shown to overlap with another fraction Cer [NH] with Cer [NH] being the major component [41]. Both Cer [AS] and Cer [NH] can only be separated after acetylation [41]. Most recently, a new ceramide class Cer [EOP] was identified [35]. Up to date, the function of each ceramide class is not known. However, the more lipophilic ceramides with ω esterified fatty acids and predominantly Cer [EOS] are reported to be of considerable importance [5].

The stratum corneum ceramides originate mainly from the deglucosylation of glucosylated ceramide precursors [17] catalyzed by the β-glucocerebrosidase [21] Another pathway is the hydrolysis of sphingomyelin by means of the acid sphingomyelinase [17, 42]. Different skin diseases such as atopic dermatitis and psoriasis were reported to affect the qualitative and quantitative pattern of stratum corneum lipids, particularly the ceramides [9, 22, 30, 31, 47], both in the involved as well as in the uninvolved skin. However, a direct relationship between both diseases and the impaired lipid profiles could not be established. The reason for that might lay in the complexity and the diversity of factors influencing the stratum corneum lipid composition such as interindividual variations [32], season [39], age [15], gender [8] and not at least the variety of the different extraction- and analytical methods used by the different investigators. On the other hand, many molecular aspects of the mentioned diseases are still unclear and need to be explored in more detail, which is best done by means of powerful analytical methods.

Previous analytical studies relied on thin-layer chromatography based on various manual procedures to separate the stratum corneum ceramides [34, 46]. Automated multiple development-HPTLC (AMD-HPTLC) has facilitated separation efficiency, quantification and reliability [4, 12, 48]. Further characterization of the ceramide classes with respect to the chain lengths of fatty acids and sphingoid bases became accessible with the use of mass spectrometry (MS). Offline coupling approaches of HPTLC and ESI-MS or ESI-MS/MS as well as reversed phase HPLC/MS were reported [36, 37, 43]. Recently, normal phase HPLC coupled to atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) was successfully used for the separation and the sensitive detection of all stratum corneum ceramides in one run [13]. Thus, the mass spectrometric profiles within each ceramide class could be directly and easily investigated. In this study, we used this new analytical approach as well as a previously developed AMD-HPTLC method [12] to investigate the extent of possible alterations regarding the ceramide profiles in the uninvolved skin by atopic dermatitis and psoriasis patients and to evaluate the feasibility of using such alterations for diagnostic purposes.

Materials and methods

Chemicals and reagents

Cer [NP] and Cer [AP] were provided by Cosmoferm (now Goldschmidt, Essen, Germany), Cer [NS] by Sederma (Le Perray en Yvelines, France), Cer [AS] (hydroxy fatty acid ceramide), cholesterol and palmitic acid were purchased from Sigma-Aldrich (Taufkirchen, Germany). These lipids were used as standards for AMD-HPTLC and HPLC/APCI-MS.

Solvents for extraction, HPTLC and HPLC/MS purposes were of HPLC grade and provided from Baker (Deventer, The Netherlands), Merck (Darmstadt, Germany) and Roth (Karlsruhe, Germany).

Subjects

The study design was permitted by the ethics committee of the medicine faculty at the Martin-Luther-University Halle-Wittenberg (Germany) and was carried out by an experienced dermatologist. After written approval was signed from all volunteers lipid extracts were obtained in vivo from 20 subjects (14 women and 6 men with an average age of 40±13 years, distributed as following: seven healthy subjects, seven atopic dermatitis- and six psoriasis patients). The inclusion criteria were an eczema area and severity index (EASI) Score>15 for atopic dermatitis patients [6, 18] and a psoriasis area and severity index (PASI)>20 for psoriasis patients [14]. Systemic as well as topical application of drugs or skin care products were not allowed 8 and 4 weeks prior to investigations, respectively.

Extraction procedure

By the in vivo surface extraction, a cylindrical glass beaker with 4 cm ID (extraction area 12.56 cm2) was filled with 10 ml n-hexane/ethanol 2:1 (v/v). The open side was pressed tightly to a skin area at the uninvolved or healthy inner forearm to prevent lateral leakage. The glass beaker was held by a PVC holder and an adjustable belt. The extraction time was exactly 5 min throughout. The extraction was carried out two times on each forearm resulting in 4 extracts per one volunteer. The extracts belonging to each volunteer were pooled and the solvents were evaporated at 50°C under a stream of nitrogen resulting in a dried residue, which thereafter was stored at −80°C. Before use, the lipid extracts were separated from hydrophilic substances and proteins as described elsewhere in detail [40]. Briefly, methanol, chloroform and distilled water were added to the extracts in the ratio 2:1:0.8 (v/v/v) leading to a homogenous solution. Upon changing the ratio to 2:2:1.8 (v/v/v), two phases were created with the stratum corneum lipids being in the lower chloroform phase, which was used for both the AMD-HPTLC- as well as for the HPLC/APCI-MS analysis.

Ceramide separation by AMD-HPTLC

The HPTLC plates were washed thrice with chloroform/methanol 65:35 (v/v) before use. Sample application was carried out automatically using an Automatic TLC Sampler 4 (Camag, Muttenz, Switzerland) on a start line located 8 mm from the bottom of the plate and at a dosage speed of 100 μl/s.

Sixteen samples were applied in lanes on each plate, including eight lanes for stratum corneum lipid extracts (each sample was analyzed in duplicate) as well as eight lanes for reference lipids of different quantities. The band length was 8 mm with 10.6 mm distance to the neighbouring lane. The development of the plates was carried out automatically using an AMD-2 apparatus (Camag, Muttenz, Switzerland) as described previously in detail [12]. Briefly, an AMD procedure including 17 steps and based on 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.

Densitometric profiling of the ceramides

After drying, the HPTLC plates were dipped into an aqueous solution of 10% CuSO4, 8% H3PO4 (v/v), and 5% methanol for 20 s. Afterwards, the plates were charred in a drying oven at 160°C for 30 min.

The developed and visualized plates were scanned from 7 mm to front using a TLC Scanner 3 (Camag, Muttenz, Switzerland). The measurements were performed in reflectance mode at a wavelength of 546 nm. The slit dimensions were 4×0.2 mm at a scan speed of 20 mm/s and a data resolution of 100 μm per step. Integration and quantification based on peak areas were performed using CATS software (Camag, Muttenz, Switzerland). To avoid experimental errors, individual curves were set up for each HPTLC plate. Quantitative results for all ceramides were related to Cer [NP] as a standard. The results were expressed as mean ± SD. For statistical analysis Student’s t-test was performed. A P value<0.01 was considered significant.

Ceramide separation and their profiling by HPLC/APCI-MS

All separations were carried out using a silica column LichroCart (125–4, 5 μm) filled with Si 60 Lichrospher particles (Merck, Darmstadt, Germany) in a normal-phase mode. The elutions were performed on a HPLC system consisting of a Spectra System P 4000 pump, an auto sampler AS 3000, and a controller SN 4000 (Thermo Separations, San Jose, CA, USA) at a flow rate of 1 ml/min and with a gradient, which is described elsewhere in detail [13].

As detector, an ion trap mass spectrometer Finnigan LCQ classic (Thermo Electron, San Jose, CA, USA) equipped with an APCI source was used throughout. The APCI heater was set to 500°C and the heated capillary to 150°C. Nitrogen was used as both auxiliary and sheath gas at flow rates of 9 and 1.2 l/min.

In all experiments, the entire flow from the HPLC was directed to the APCI source. The injected ceramide amount was 5 μg for each sample (including all ceramide classes). The spectra of ceramides were obtained in full scan mode between m/z 450 and 1500.

Results

Ceramide profile as obtained by AMD-HPTLC and densitometry

The extracted lipids were analyzed by AMD-HPTLC leading to the separation of the ceramides from other lipids and the ceramides themselves into seven fractions. After densitometric evaluation, the total ceramide amount, expressed in μg/cm2, as well as the percentage profile were determined for each sample. From Table 1, it can be observed that the ceramide amount in the uninvolved skin of atopic dermatitis (15.3±4.0 μg/cm2) and psoriasis (11.4±3.5 μg/cm2) patients is reduced when compared to healthy skin (16.2±3.5 μg/cm2) with the reduction being more pronounced in the psoriasis patients .The differences were however not significant according to the performed Student’s t test at P<0.01. In Table 2 and Fig. 1, the percentage composition of the ceramide classes are presented. Again, no significant changes could be observed. These results indicate that the ceramide composition of the uninvolved skin in atopic dermatitis and psoriasis patients is not affected and comparable with that in healthy skin.

Table 1 Amount of ceramides per extraction area
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Table 2 Ceramide profile of healthy controls versus uninvolved skin of atopic dermatitis and psoriasis patients as determined by AMD-HPTLC and densitometry
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Fig. 1
figure 1

Ceramide profile after densitometric evaluation. No significant differences were obtained. (P<0.01, healthy: n=7, dermatitis: n=7 and psoriasis: n=6)

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This study focuses clearly on ceramides. This is due to their utmost importance for the barrier function as well as because of their structural variability. In addition, Table 3 shows data for other lipid classes. Significant differences could not be obtained neither for cholesterol nor for free fatty acids.

Table 3 Composition of stratum corneum main lipids in healthy skin compared to uninvolved skin in atopic dermatitis and psoriasis patients as analyzed by AMD-HPTLC and densitometry
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Ceramide profile by means of normal phase HPLC/APCI-MS

In addition to the densitometric data, the chain length profile within each ceramide class is also of importance. It namely allows examining possible alterations at the molecular level.

This requires a specific MS detection. Therefore, a recently developed HPLC/APCI-MS [13] method for the separation and on line detection of all stratum corneum ceramides in one run was carried out. The mass spectra relating to the ceramides of the uninvolved regions of patients’ skin were thereafter compared to those of healthy skin. As a result, no significant differences between healthy and uninvolved skin in both diseases could be obtained.

In Fig. 2, the ceramide separation chromatograms for the categories: healthy, atopic, and psoriatic volunteer is shown. In Figs. 3, 4 and 5 the mass spectrometric profiles of selected ceramides corresponding to the same samples presented in Fig. 2 are demonstrated. As can be observed, differences between healthy and diseased persons could not be obtained, neither regarding the separation pattern nor concerning the mass spectrometric profiles of the presented ceramides [EOS], [NP], and [NH], which were reported to be altered by atopic dermatitis and psoriasis. The same result was also obtained by all other ceramide classes, which are not illustrated here.

Fig. 2
figure 2

Separation of stratum corneum ceramides by means of normal phase HPLC/APCI-MS; a, b and c correspond to the ceramide separation by a healthy volunteer, an atopic dermatitis and a psoriasis patient, respectively. In all chromatograms, 1 Cer [EOS], 2 Cer [NS], 3 Cer [NP], 4 Cer [EOH], 5 Cer [NH] and Cer [AS], 6 Cer [AP], 7 Cer [AH]

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Fig. 3
figure 3

Mass spectra [M − H2O + H]+ of Cer [EOS] by a healthy volunteer, b atopic dermatitis patient and c psoriasis patient. The spectra correspond to peak 1 in Fig. 2

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Fig. 4
figure 4

Mass spectra [M + H]+ of Cer [NP] by a healthy volunteer, b atopic dermatitis patient and c psoriasis patient. The spectra correspond to peak 3 in Fig. 2

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Fig. 5
figure 5

Mass spectra [M –H2O+ H]+ of Cer [NH] by a healthy volunteer, b atopic dermatitis patient and c psoriasis patient. The spectra correspond to peak 5 in Fig. 2

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Discussion

To achieve precise and reliable results, only patients fulfilling the inclusion criteria of an EASI>15 or a PASI>20 were considered. Such criteria would reveal significant results, if ceramide alterations were manifested in the uninvolved skin of atopic dermatitis or psoriasis patients. In addition, the extractions were performed by experienced dermatologists. By choosing the extraction mixture both the compliance of the patients as well as the extraction efficiency were considered. Although various extraction procedures may expose different results, it has to be taken into account that the relative amount of the ceramide classes do not change with stratum corneum depth [44]. The levels of ceramides, cholesterol and the free fatty acids were reported to be about 60%, 18% and 22%, respectively [44]. This finding is consistent with our results (Table 3).

In this study, we have used both AMD-HPTLC [12] with densitometric detection and normal phase HPLC/APCI-MS [13] to analyze the extracted stratum corneum lipids, and particularly the ceramides. The used AMD-HPTLC procedure has the advantages not only to enable the separations of all stratum corneum lipids in one run in a relatively short time but also to reveal consistent quantifications due to compressed thin bands resulting from the several gradient steps. With the utilized HPLC/APCI-MS method, it was possible to separate and profile the stratum corneum ceramide classes simultaneously and thus to facilitate studying probable ceramide alterations at the molecular level. Furthermore, it is to our knowledge the first time, in which a direct mass spectrometric analysis of the distinctive lipophilic and for ESI-MS not accessible ceramides such as Cer [EOS] was performed.

Regarding atopic dermatitis, it was reported that alterations in the total and relative amounts of the ceramides represent an etiologic factor for the disease, Cer [EOS] being most affected both in the involved and the uninvolved skin [22]. Bleck et al. showed that in non-lesional (uninvolved) skin of atopic dermatitis patients, Cer [AS] consists of two subfractions, which can be separated by HPTLC [3]. In addition, a reduction of Cer [NP] was found to correlate with an increased transepidermal water loss (TEWL) in both involved as well as uninvolved skin [9]. A more recent work showed a decrease of Cer [EOH] by atopic dermatitis patients [27]. On the biochemical level, the enzymes sphingomyelin deacylase [19], glucosylceramide deacylase [23] as well as a bacterial derived ceramidase [33] were recently reported to be possible causes for the ceramide deficiency. Conversely, the activities of the sphingolipid biosynthesis enzymes β-glucocerebrosidase [24] and acid sphingomyelinase [26], were found to be not essentially impaired.

Di Nardo et al. [9] have defined a “clinically and functionally in-between population” of atopic dermatitis patients, who show intermediate levels of lipids. A study performed by Matsumoto et al. revealed that the decrease in the total ceramide amount and in Cer [EOS] is restricted to the involved atopic skin and is not extended to the uninvolved areas [29]. Furthermore, patients free form atopic dermatitis symptoms for more than 5 years showed normal TEWL and water content values [28]. Our data are consistent with these findings. Neither the total absolute (Table 1) or the relative (Table 2, Fig. 1) ceramide amounts nor the mass spectrometric profiles of Cer [EOS] (Fig. 3), Cer [NP] (Fig. 4), Cer [NH] (Fig. 5) in the uninvolved skin of atopic dermatitis patients prove any significant differences compared to healthy controls. Similar results regarding the mass spectra of Cer [NS], [EOH], [AP], [AH] were obtained (Data not shown).

In psoriatic skin Cer [EOS], [NP], [EOH], [AS], [AP] were reported to be decreased and Cer [NS] to be increased. The decrease of Cer [EOS] and Cer [EOH] were suggested to be responsible for the defective barrier function [30, 31]. Others [1] have found that the level of prosaposin, an activator protein in the sphingolipid pathway, is decreased in psoriasis patients, the decrease being more pronounced in the involved skin compared to the uninvolved skin. On the other hand, it was previously found that TEWL and water content do not differ in nonlesional (uninvolved) skin of psoriatic patients when compared to control subjects [2] indicating an intact barrier function. The results obtained here support this finding.

From this study, we conclude that the uninvolved skin of atopic dermatis or psoriasis patients can be considered to be “healthy” regarding the composition of the ceramides and the other investigated lipids. Consequently, the free extractable stratum corneum lipids, and particularly the ceramides in the uninvolved skin of the mentioned diseases cannot be used for diagnostic purposes. In addition, our findings imply that impairments in the composition of the stratum corneum lipids and thus in their biosynthesis do not represent an obligate etiologic factor neither for atopic dermatitis nor for psoriasis. However, Ceramide profiles of different extraction procedures and from different anatomical sites are to be compared. Moreover, further investigations by means of tandem MS are to be carried out. In Addition, one has to bear in mind that the barrier function is not restricted to the intercellular lipids and primarily ceramides but also includes other components such as the cornified envelope, the lipid cornified envelope, the corneocytes and the lipid organization, which can be affected in skin diseases and therefore have also to be taken into account.