Characterization of overall ceramide species in human stratum corneum.

Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and water-holding functions of the skin, and specific species might be closely related to the regulation of keratinization, together with other CER-related lipids. Structures of those diverse CER species, however, have not been comprehensively revealed. The aim of this study was to characterize overall CER species in the SC. First, we constructed 3D multi-mass chromatograms of the overall CER species, based on normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile salt-containing polar solvent. The CERs targeted from the 3D chromatograms were structurally analyzed using NPLC-ESI-tandem mass spectrometry (MS/MS), which resulted in the identification of 342 CER species in the inner forearm SC. This led to the discovery of a new CER class consisting of alpha-hydroxy fatty acid and dihydrosphingosine moieties, in addition to the 10 classes generally known. The results also revealed that those CERs contain long-chain (more than C(18))-containing sphingoids and a great number of isobaric species. These novel results will contribute not only to physiochemical research on CERs in the SC but also to lipidomics approaches to CERs in the skin.

Ceramides (CERs) not only have physicochemical roles as a barrier against cell permeability and as a matrix for the association of membrane proteins but also have physiological roles in signal transduction and cell regulation relevant to apoptosis, cell growth arrest, differentiation, senescence, and immune responses (1)(2)(3)(4). Compared with other human cells/tissues, the stratum corneum (SC) of human skin has extremely complex CERs consisting of fatty acid moieties (nonhydroxy, a-hydroxy, or ester-linked v-hydroxy) and sphingoid moieties (sphingosine, dihydrosphingosine, phytosphingosine, or 6-hydroxy-sphingosine) (5). CERs in human SC contribute to the formation of lamellae structures at intercellular spaces among the horny cells in the SC together with cholesterol and free fatty acids and play important physicochemical roles in determining cutaneous barrier and water-holding functions (6)(7)(8)(9). On the other hand, some scientists are interested in the dynamics of CER-related lipids in differentiated keratinocytes, where apoptosis occurs in the skin (10). In recent years, the possibility that specific CER species may be involved with human diseases and/or with physiological phenomena has also been proposed (11)(12)(13). This suggests that specific CER species might be closely related to the regulation of keratinization from living epidermal cells to dead horny cells, together with other CER-related lipids. However, until now, structures of CER species present in the SC have not been comprehensively revealed.
For human cells/tissues, except for hair and SC, there are 3 classes (combinations of nonhydroxy fatty acids and sphingosines, nonhydroxy fatty acids and dihydrosphingosines, and a-hydroxy fatty acids and sphingosines) that contain simple species with combinations of C 18 -containing sphingosines or dihydrosphingosines and even carboncontaining nonhydroxy or a-hydroxy fatty acids (13)(14)(15). Human hair CERs are more complex than are those in human cells/tissues, because hair contains 4 classes (combinations of nonhydroxy fatty acids and sphingosines, nonhydroxy fatty acids and dihydrosphingosines, a-hydroxy fatty acids and sphingosines, and a-hydroxy fatty acids and dihydrosphingosines), and the predominant class, CERs consisting of nonhydroxy fatty acid and dihydrosphingosine moieties, has C 16 -, C 17 -, C 18 -, C 19 -, or C 20 -containing dihydrosphingosines and not only even but also odd carbon-containing nonhydroxy fatty acids (16). On the other hand, human SC has even more-complex CERs. Thus, from earlier days when CERs were analyzed using TLC, it was recognized that in the SC there are 7 CER classes corresponding to seven spots on the TLC plates (17,18). Robson et al. (5) and Vietzke et al. (19) demonstrated that the seven spots correspond to 9 CER classes, because five of the spots come from single CER classes (combinations of nonhydroxy fatty acids and phytosphingosines, nonhydroxy fatty acids and 6-hydroxy-sphingosines, a-hydroxy fatty acids and phytosphingosines, a-hydroxy fatty acids and 6-hydroxysphingosines, and ester-linked v-hydroxy fatty acids and sphingosines), whereas two spots contain 2 CER classes (combinations of nonhydroxy fatty acids and dihydrosphingosines, nonhydroxy fatty acids and sphingosines, a-hydroxy fatty acids and sphingosines, and ester-linked v-hydroxy fatty acids and 6-hydroxy-sphingosines). After that, an additional class, CERs consisting of v-hydroxy fatty acid and phytosphingosine moieties, was discovered (20), which seemed to have been not recognized earlier due to its trace levels in the SC. Therefore, in addition to the 2 classes of covalently bound types (5,21), 10 classes have been known as free extractable types in the SC. A few reports have described combinations of fatty acid and sphingoid moieties in CER species in the SC, such as those by Vietzke et al. (19) and Farwanah et al. (22). However, those descriptions were based on the assumption that all sphingoids have 18 carbons. On the other hand, Hsu et al. (23) did structural studies of CER species in the SC. However, their structural analysis was restricted to a part of CERs that have shorter chain lengths in the sphingoid moieties.
The aim of this study was to characterize overall CER species in human SC. Normal-phase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) was optimized for comprehensive profiling of diverse CERs in the SC, and overall CER species were visualized as 3D multi-mass chromatograms, followed by the structural analysis of the targeted peaks in the 3D chromatograms by NPLC-ESI-tandem mass spectrometry (MS/MS). Such analysis has also uncovered a new CER class consisting of a-hydroxy fatty acid and dihydrosphingosine moieties. Herein, we describe the results of identification of CERs in the SC, which are as many as 342 species.

Nomenclature
Classes of CERs are termed according to previous reports (5,24). Briefly, nonhydroxy fatty acid, a-hydroxy fatty acid, esterlinked nonhydroxy fatty acid, v-hydroxy fatty acid, dihydrosphingosine, sphingosine, phytosphingosine, and 6-hydroxy-sphingosine moieties are designated as N, A, E, O, DS, S, P, and H, respectively. Based on this terminology, 12   Characterization of ceramides in stratum corneum 1467 N, A, E, or O in parentheses, and the number of sphingoid carbons is expressed following the letters DS, S, P, or H in parentheses, as represented in CER[E(18:2)O(30)H (20)] for a molecule consisting of ester-linked linoleic acid, v-hydroxy triacontanoic acid, and C 20 6-hydroxy-sphingosine moieties.

Chemicals
Methanol, 2-propanol, chloroform, and n-hexane of HPLC grade from Kanto Reagents (Tokyo, Japan) were used to prepare extracted SC lipid solutions and mobile phases for NPLC. Ultra-pure water prepared using a Milli-Q purification system (Millipore; Bedford, MA) was used in all procedures. All other chemicals were of the highest analytical grade commercially available. Twelve authentic materials, CER[N(26)DS (20) (20)], were synthesized in our laboratory. Those authentic CERs were synthesized by coupling corresponding fatty acids and sphingoids in the presence of 1-ethyl-3-(3-diethylaminopropyl)carbodiimide hydrochloride, with 1-hydroxybenzotriazole as the coupling reagent (25). Constitutional fatty acids, except for hexacosanoic acid and linoleic acid, and sphingoids were prepared according to the literature (26)(27)(28)(29)(30)(31). Each of the authentic CERs synthesized was purified with flash silica-gel column chromatography and/or recrystallization. Analyses by 1 H-NMR, 13 C-NMR, and infrared spectometry confirmed the structures (see supplementary  Table I), and the purities were checked using TLC and LCultraviolet detection (.95%). Each authentic solution was prepared in a given molar solution. Individual authentic stock solutions (1 mM each) were prepared by dissolving accurate amounts of the authentic CERs in chloroform-methanol (1:1; v/v) and were stored at 24°C. When individual or mixed working authentic solutions were obtained, aliquots of the stock solutions were dried using a nitrogen stream, and the residues were diluted with n-hexane-2-propanol-formic acid (95:5:0.1; v/v/v) just prior to use.

Materials
The protocol was approved by the Ethical Committee of the Kao Corporation of Japan, based on recommendations from the Declaration of Helsinki. Tape-stripped SC specimens were collected from six healthy Japanese volunteers (three males 26 to 42 years old and three females 25 to 38 years old, who were enrolled in the study with informed consent) and were used to prepare extracted lipid materials, followed by the profiling of CERs using NPLC-ESI-MS. To obtain SC specimens, a polyphenylene sulfide film tape (Nichiban; Tokyo, Japan) with an area of 18 mm 3 25 mm was pressed to the skin at the inner forearm and was then stripped. When 3 or 10 consecutive tapes were collected, they were pressed to the same region of the skin as the first one. The 3 or 10 tapes were immersed in methanol with sonication for 10 min. The lipid extracts were dried using a nitrogen stream and were then dissolved in chloroformmethanol (99.5:0.5; v/v). This lipid solution was applied to a Sep-Pak Vac RC silica cartridge (Waters; Milford, MA) that had been conditioned by chloroform-methanol (99.5:0.5; v/v), followed by solid-phase extraction with 10 ml chloroform-methanol (99.5:0.5; v/v) and 10 ml chloroform-methanol (99:5; v/v). The latter fraction, which contains the CERs, was dried using a nitrogen stream and then dissolved in n-hexane-2-propanol-formic acid (95:5:0.1; v/v/v). To identify CERs in the inner forearm SC, the extracted lipid samples obtained from a 34-year-old male and a 26-year-old female with 10 consecutive tapes were mixed and then subjected to NPLC-ESI-MS/MS. Amounts of lipid extracts from the 3 and 10 tapes were approximately 15 and 50 mg, respectively.

Analysis using NPLC-ESI-MS
An Agilent 1100 Series LC/MSD SL (single quadrupole) system equipped with an ESI source, ChemStation software, an 1100-well plate autosampler (Agilent Technologies; Palo Alto, CA), and an Inertsil SIL 100A-3, 1.5 mm i.d. 3 150 mm column (GL Science; Tokyo, Japan) was used. In this LC/MSD system, an 1100 binary pump was connected to two mobile phases [A: n-hexane-2-propanol-formic acid (95:5:0.1; v/v/v); and B: nhexane-2-propanol-50 mM ammonium formate aqueous solution (25:65:10; v/v/v)] with a flow rate of 0.1 ml/min. The mobile phases were consecutively programmed as follows: a linear gradient of A 100-90% (B 0-10%) between 0 and 3 min, a linear gradient of A 90-0% (B 10-100%) between 3 and 35 min, an isocratic elution of A 0% (B 100%) for 5 min, a linear gradient of A 0-100% (B 100-0%) between 40 and 50 min, and an isocratic elution of A 100% (B 0%) from 50 to 80 min for column equilibrium (a total run time of 80 min). The injection volume was 10 ml each of authentic solutions or extracted SC lipid solutions. The column temperature was maintained at 40°C. To enhance ionization of molecular-related ions derived from CERs in the mass spectrometer, 2.5 mM ammonium formate 2-propanolmethanol (50:50; v/v) at a flow rate of 0.1 ml/min was added to the mobile phase at the postcolumn position via a T-connector. Parameters for scan measurement of ESI using unit mass resolution mode was finally determined in the following settings: polarity, positive; heater temperature of nitrogen gas, 300°C; flow of heated dry nitrogen gas, 8.0 l/min; nebulizer gas pressure, 20 psi; capillary voltage, 3,500 V; fragmenter voltage, 150 V; scan range, 250-1,500 amu; scan time, 1.9 s/cycle.

Optimization of NPLC-ESI-MS conditions
To analyze CERs in human SC using LC-MS, we decided to use NP and ESI modes in LC and MS, respectively, because the NP mode may enable the class separation of diverse CERs that have chain distributions both in the fatty acid and sphingoid moieties based on differences in their polarities, whereas the ESI mode seems to be the best for the LC-MS analysis of CERs, as demonstrated in many analytical studies (14,15,(32)(33)(34)(35)(36).
First, the NPLC separation was optimized using 12 kinds of authentic materials, CER[N(26)DS (20) for all authentics in the negative-ion mode. Therefore, the combination of positive-ion ESI and postaddition of ammonium formate-containing 2-propanol-methanol solution following column separation was selected as the optimal condition for the detection of diverse CERs by MS. The concentration of ammonium formate was optimal at 2.5 mM in terms of signal to noise. MS parameters, such as heater temperature of nitrogen gas, flow of heated dry nitrogen gas, nebulizer gas pressure, capillary voltage, and fragmenter voltage, were optimized to achieve higher abundances of the targeted molecular-related ions for the 12 authentic CERs under positive-ion ESI-MS conditions (data not shown).   20)]. This indicated that the mass spectral patterns are dependent on the types of sphingoid moieties, that is, the hydrophilic structures, under the less-polar mobile phase system. Thus, there seemed to be three types:

Comprehensive profiling of CERs in human SC
The optimized NPLC-ESI-MS technique was then applied to the analysis of CERs in the SC. Fig. 3. shows the TIC for crude extracted lipids from three consecutive SC-stripped tapes of the inner forearm of a 34-year-old male. It was supposed that the CERs would elute at ?6 to 19 min under these chromatographic conditions, based on the retention times of authentic CERs, whereas large peaks eluting at ?20 min were judged to be contamination from the tapes used, because they appeared in blank analyses without any stripped SC. At an early stage of this study, on the basis of the characteristics of mass spectra of all peaks detected, we tried to observe overall CERs that are composed of diverse chain distributions both in fatty acid and sphingoid moieties, similar to a procedure reported by Farwanah et al. (22,37). However, comprehensive observation was impossible, especially in the case of extremely low-abundance species, because m/z values corresponding to those species were often not found, due to the influence of high-content species.
To resolve this problem, we constructed 3D multi-mass chromatograms of the CERs, consisting of retention time (X-axis), m/z (Y-axis), and intensity (Z-axis), as presented in Fig. 4, which were constructed by extraction of 200 higher intensive ions in the range of retention times between 4 and 22 min from TIC of Fig. 3. The assignment of total carbons and unsaturation (if present) was performed as follows. i) We examined whether there were peaks that substantially coincided with retention times and m/z values of molecular-related ions derived from the 12 authentic CERs. ii) Other candidates, which differed from the identified CERs (i.e., authentic ones) at intervals of every carbon unit (14 as amu), were explored. In this survey, a regularly linear pattern was noted, in which retention times of CER species with higher molecular weights were slightly shorter than those with lower molecular weights under the NPLC conditions, in common with each class (Fig. 4). iii) As other possibilities, candidates that have differences of unsaturated degrees in N, A, E, or O moieties were also explored based on mass number gaps of 2 amu. iv) If there were no peaks corresponding to an authentic CER and no peaks were found in the neighborhood, a CER class was judged to be absent in the SC.
In the inner forearm SC of a 34-year-old male, the peaks were assigned as CER  (Fig. 4). No species of CER[EODS] or with unsaturation in N, A, or O moieties were found. Similarly, we analyzed and assigned the CERs in the inner forearm SC of five other subjects (two males and three females). Consequently, the 3D multi-mass chromatograms and their assignment were almost the same as those of the 34-year-old male (data not shown). Thus, 11 CER classes were present in the SC tested; no species of CER[EODS] and none with unsaturated fatty acid moieties (except for E) were found. There were only differences in small peaks. A list of the commonly observed CERs in all subjects is shown in Table 1. This indicates that at least in the inner forearm SC of the six healthy subjects tested, N-containing CERs contain molecules with larger total carbons than do A-containing CERs. Also, EO-containing CERs had relatively narrow chain length distributions, compared with the other CERs.

Characteristics of product ion spectra of authentic CERs
The 12 authentic CERs were applied to NPLC-ESI-MS/ MS to characterize their product ion spectra under the optimized NPLC separation conditions using the low polar mobile phases.   These results indicated that product ions obtained in the positive-ion mode can provide information on the number of carbons of sphingoid moieties and those obtained in the negative-ion mode can identify the number of carbons of fatty acid moieties. The total carbons of CERs in the inner forearm SC were assigned already, as shown in Fig. 4 and Table 1. To characterize combinations of fatty acid and sphingoid moieties for the CERs detected, we decided to use one of the prominent product ions for each of the positive-and negative-ion modes in NPLC-ESI-MS/ MS, except for two product ions in the negative-ion mode for the identification of the EO-containing CERs, as shown in Table 2. Although identification using at least three diagnostic ions in a product ion spectrum would be desired for precise structural analysis using LC-MS/MS (16,41), we decided to use only two diagnostic product ions (each derived from the positive and negative spectra) for N-or A-containing CERs, because we could detect prominent product ions derived from extremely low level CERs in actual crude lipid samples of SC but the corresponding less-abundant product ions could not be substantially detected due to the low detection sensitivity of the MS/MS scan analysis. However, in this study, the retention times greatly helped us to identify the individual CERs, in addition to the precursor/product ions arising from two different detection modes. As already described, the retention times obtained from this NPLC system were quite informative, because there was a regularly linear pattern for each of the CER classes commonly, under the optimized NPLC conditions (Fig. 4). Therefore, we supposed that reliable identification would be possible based on the evidence of the two diagnostic ions and the retention times. Figure 5 shows representative positive and negative product ion spectra of CER[NS] with 49 total carbons extracted from the inner forearm SC. In the positive product ion spectra, an ion at m/z 292 (ion #1 in Fig. 5 Table 2)    peaks of the CERs assigned in the NPLC-ESI-MS analysis (Fig. 4, Table 2) were subjected to the structural analysis. When peaks that were commonly observed in all six subjects (Table 1) supplementary Table II). Those CERs contained the great number of isobaric species that have identical molecular weights but different combinations of fatty acid and sphingoid moieties, as shown in the case of Fig. 5, which depicts six isobaric CER[NS] species having 49 total carbons.

DISCUSSION
In this study, as authentic materials, we used eight CERs having 46 total carbons that consist of 26 and 20 carbons in the fatty acid and sphingoid moieties, respectively, for N-or A-containing CERs, and four CERs having 68 total carbons consisting of 18, 30, and 20 carbons in E, O, and sphingoids, respectively, for EO-containing CERs. To comprehensively examine diverse CERs in the SC, it would be ideal to use various authentic materials of different chain lengths. However, it seemed impossible to synthesize all such materials when realistic tasks that would require very problematic and time-consuming labor were considered. Therefore, we adopted another strategy: we would use an authentic material representative of each CER class. As a result, in our preliminary survey on CERs in the SC, it was revealed that CER species having 46 total carbons that consist of 26 and 20 carbons in the fatty acid and sphingoid moieties, respectively, seem to be commonly present and to be almost the center of the carbon length distributions in all N-or A-containing CER classes. Also, CER species having 68 total carbons, consisting of 18:2, 30, and 20 carbons in E, O, and sphingoid moieties, respectively, seemed to be common in all EO-containing CER classes except for CER [EODS]. Therefore, using 12 such authentic materials, we optimized NPLC-ESI-MS conditions and identified overall CER species present in the SC.
First, we optimized the NPLC conditions within 20 min, using a gradient elution system, which allowed the analysis of all CER species belonging to the 11 classes of CERs. One of the most ideal characteristics of this NPLC system is that different CER species belonging to the same class have regularly linear patterns in the chromatograms, in which CER species with higher molecular weights elute slightly earlier than those with lower molecular weights. This is extremely helpful in the practical analysis of CERs in SC specimens, because one can tentatively identify their total carbons using only their retention times and m/z values without a burdensome MS/MS analysis. Next, based on examinations of detection sensitivity, the postcolumn addition of a polar solvent, together with a volatile salt [2.5 mM ammonium formate-containing 2-propanolmethanol (50:50; v/v)] via a T-connection, is employed. This drastically enhances the intensities of molecularrelated ions for all authentic CERs belonging to different classes. The drastic enhancement of detection leads to 3D visualization of CER profiles of the SC by their multi-mass chromatograms, consisting of retention time (X-axis), m/z (Y-axis), and intensity (Z-axis). Without such improvements in both separation and detection following such a 3D visualization, we would not have succeeded in the identification of overall CER species.
We had already developed a reverse-phase LC-MS method for CERs in hair (16). That method has an advantage in detection sensitivity compared with the NPLC-ESI- MS method. However, that method is not appropriate currently to analyze overall CERs in the SC, because extremely hydrophobic CER species, such as EO-containing CERs, are not eluted under those reverse-phase conditions. To get peaks of the hydrophobic CERs, we could modify the analytical conditions by use of a more lowpolar solvent as one of mobile phases in a reverse-phase gradient system. However, even if we got those peaks under reverse-phase conditions, the chromatogram would be too complicated to tentatively identify peaks of overall CERs due to complex separation based on both hydrophobic and hydrophilic degrees. In contrast to this, when the NPLC-ESI-MS method is used for the analysis of overall CERs in the SC, their tentative identification can be readily performed because the regularly linear patterns are available as mentioned above. Therefore, we believe that NPLC-ESI-MS is obviously superior to reverse-phase LC-MS from the standpoint of characterization of extremely diverse CERs in the SC, although detection sensitivity is lower. The comprehensive profiling using NPLC-ESI-MS is followed by a comprehensive identification of diverse It should be kept in mind that analytically targeted peaks were restricted to those commonly observed in all six subjects tested, and that more than 342 CER species would have been identified in the SC if the analytical conditions had had a higher detection sensitivity. However, it seems that the major species of CERs have all been identified. We observed no differences in the 3D chromatographic patterns of CER species providing relatively large peaks between individuals tested, although there were differences in those providing relatively small peaks (data not shown). In conclusion, we successfully achieved comprehensive identification of CERs, which number as many as at least 342 species, under the optimized NPLC-ESI-MS/ MS conditions.
Here, we consider novel aspects regarding the structures of CER species in the SC. This study clarified combinations of constitutional fatty acids and sphingoids that comprise 342 distinct CER species. Although a few reports have described combinations of fatty acid and sphingoid moieties in CER species (19,22,23), our study is substantially the first report of the precise identification of overall CER species in the SC, because the previous reports were based on the assumption that all sphingoids have 18 carbons (19,22), or were restricted to CERs that have shorter chain lengths in their sphingoid moieties (23). The validity of our results can be estimated by comparison with a previous report by Wertz et al. (42), who used GC with derivatizing techniques and then analyzed both the fatty acid and sphingoid moieties that had been obtained from the hydrolysis of each of the isolated CER classes.  (42) found that linoleic acid was the most predominant, although palmitic acid, stearic acid, and oleic acid were also present. As for the number of carbons of the DS, S, or P moiety, although our results are nearly the same as those of the earlier report (42), our study reveals a slightly greater number of carbons, presented as C 16-28 DS in CER[NDS] and C [16][17][18][19][20][21][22][23][24][25][26] S in CER[NS], compared with the previous result, C [16][17][18][19][20][21][22] in both classes (42). Those slight differences might be derived from distinct origins, including inter-individual variations, or from other factors, such as experimental artifacts during the preparative procedures or by GC separation performance. Nonetheless, the differences are only slight, and we believe that our identification results are close to the previous report (42). Some scientists may believe that C 18 -containing sphingoids are predominant in the SC (19,22), but our study demonstrates that there are many CER species with long-chain (more than C 18 )containing sphingoids in the SC.
When compared with CERs in other human cells/tissues, including hair, it is evident that CERs in the SC are characterized by the diversity of CER classes, as is generally known. In addition, this study indicates that another characteristic of CERs in the SC is that the species have longer chain lengths, especially in sphingoids, than do those of other cells/tissues, including hair. This is exemplified by the facts that CER [NS] in the SC is composed of C 16-30 N and C 16-26 S moieties, whereas CER[NS] in other cells/ tissues (except for hair) is composed of C 16-26 N and C 18 S (43,44), and that CER [NDS] in the SC is composed of C 16-30 N and C 16-28 DS, whereas CER[NDS] in hair is composed of C 14-30 N and C [16][17][18][19][20]. This characteristic may be necessary to maintaining the barrier function of human skin, possibly because CER species with longer chain lengths seem to be appropriate for preventing water from diffusing from the SC to the outside, or for preventing chemicals from penetrating from the outside to the SC. Regarding the comparison between CERs in the SC and hair, there are similarities and differences. The similar characteristics are the large number of odd carbonscontaining CERs, some of which are assumed to have branched chains either in the fatty acid or the sphingoid moiety in human hair (16). The presence of odd carbonscontaining CERs in the SC is in agreement with the previous study by Wertz et al. (42). As is generally known, human sebum has odd carbons-containing fatty acids with branched carbon skeletons (45). Therefore, the odd carbons-containing CERs in the SC might have branched chains. This possibility should be clarified in the future by further investigations, together with synthesis of authentic CERs containing branched fatty acids and/or branched sphingoids. On the other hand, one different characteristic is that CERs in the SC have no unsaturated fatty acid moieties except for E (linoleic acid), whereas hair contains many CER species with unsaturated ones (16). Despite different and unknown characteristics, CERs in the SC are relatively similar to hair CERs, compared with other cells/ tissues. This may be due to the anatomical and developmental similarities of human skin and hair.
Currently known de novo pathways for diverse CERs in human SC are arranged as follows: 1) production of 3-ketosphinganine from palmitoyl-CoA and L-serine by serine palmitoyltransferase (46); 2) production of dihydrosphingosine from 3-ketosphinganine by 3-ketosphinganine (3-ketodihydrosphingosine) reductase (46); 3) production of a-hydroxy fatty acid from nonhydroxy fatty acid by 2-hydroxylase, designated as FA2H (47) (50). To our knowledge, the biosynthetic pathways for EO-containing and/or H-containing CERs are unknown at present. However, it can be assumed that the biosynthetic pathway of H-containing CERs would be similar to those of S-or P-containing CERs by unidentified enzymes, that is, H-containing CERs might be produced from DS-containing CERs. Taken together with our results on the identification of overall CER species in the SC, these pathways lead us to the interesting notion. Thus, according to the above pathway, C 18 -containing DS would be preferentially produced in human epidermis, followed by the occurrence of CER species with C 18 -containing sphingoids. However, we identified many species with longchain (more than C18)-containing sphingoids in the SC. This phenomenon is of considerable interest from the viewpoint of the biosynthesis of CERs in human skin. In addition, there are differences in chain length distributions of both fatty acid and sphingoid moieties among CER [ , respectively. This might suggest the chain-dependent selectivity of DEGS1, DEGS2, and unidentified H-containing CER-related enzyme(s). Further study will be required to clarify phenomena that might be related to the reasons that CER species with such extremely diverse chain length distributions are present in the SC.
In conclusion, we have structurally analyzed overall CER species in human SC using a newly constructed NPLC-ESI-MS/MS technique, followed by 3D multi-mass chromatograms. This has revealed that there are at least 342 species within 11 classes in the SC. Those CER spe-cies include a new class, CER[ADS], long-chain (more than C 18 )-containing sphingoid moieties, and a great number of isobaric species. These novel results will contribute not only to future research on the physiochemical roles of CERs at the species level in the SC but also to lipidomics approaches to CERs (and CER-related lipids) in the skin. Our next project will be to design another method for providing comprehensively quantitative levels of the 342 CER species; methods exist for the quantification of specific CER species but not for the comprehensive quantification of as many as the 342 CER species present in the SC. Such a quantitative method could be of further use in the study of the physicochemical relationships between the CER species and the various types of SC (different ages, sexes, regions, skin conditions, and so on), or in research into the lipidomics of CERs in the skin, such as clarification of the physiological roles of specific CER(s) during keratinization and of the de novo biosynthesis of diverse CER species of different chain lengths. That method should be also highly sensitive, because small amounts of crude lipids obtained from only one SC-stripped tape can be applied. That is because if such a highly sensitive method is acquired, we may even characterize depth profiles of overall CER species within the SC by analysis of each of the tapes consecutively stripped at the same SC regions.