Journal of Lipid Research Volume 49, 2008
Yoshinori Masukawa,Hirofumi Narita, Eri Shimizu, Naoki Kondo, Yoshiya Sugai, Tsuyoshi Oba, Rika Homma, Junko Ishikawa, Yutaka Takagi, Takashi Kitahara, Yoshinori Takema, and Katsumi Kita Tochigi Research Laboratories, Kao Corporation, Ichikai, Haga, Tochigi 321-3497, Japan
Abstract Ceramides (CERs) in human stratum corneum (SC) play physicochemical roles in determining barrier and waterholding 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 normalphase liquid chromatography (NPLC) connected to electrospray ionization-mass spectrometry (ESI-MS) using a gradient elution system and a postcolumn addition of a volatile saltcontaining 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 α-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 C18)-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.—Masukawa, Y., H. Narita, E. Shimizu, N. Kondo, Y. Sugai, T. Oba, R. Homma, J. Ishikawa, Y. Takagi, T. Kitahara, Y. Takema, and K. Kita. Characterization of overall ceramide species in human stratum corneum. J. Lipid Res. 2008. 49: 1466–1476.
Supplementary key words electrospray ionization tandem mass spectrometry • identification • isobaric • multi-mass chromatograms • normalphase liquid chromatography • long chain-containing sphingoid
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 (1–4). Compared with other human cells/tissues, the stratum corneum (SC) of human skin has extremely complex CERs consisting of fatty acid moieties (nonhydroxy, α-hydroxy, or ester-linked ω-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 (6–9). 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 (11–13). 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 α-hydroxy fatty acids and sphingosines) that contain simple species with combinations of C18-containing sphingosines or dihydrosphingosines and even carboncontaining nonhydroxy or α-hydroxy fatty acids (13–15). 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, α-hydroxy fatty acids and sphingosines, and α-hydroxy fatty acids and dihydrosphingosines), and the predominant class, CERs consisting of nonhydroxy fatty acid and dihydrosphingosine moieties,has C16-, C17-, C18-, C19-, or C20-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, α-hydroxy fatty acids and phytosphingosines, α-hydroxy fatty acids and 6-hydroxy-sphingosines, and ester-linked ω-hydroxy fatty acids and sphingosines), whereas two spots contain 2 CER classes (combinations of nonhydroxy fatty acids and dihydrosphingosines, nonhydroxy fatty acids and sphingosines, α-hydroxy fatty acids and sphingosines, and ester-linked ω-hydroxy fatty acids and 6-hydroxy-sphingosines). After that, an additional class, CERs consisting of ω-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 α-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.