LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Abstract

organization of these lipids are of major importance for a competent skin barrier function ( 1-4 ). The major lipid classes in human SC are cholesterol, free fatty acids, and ceramides (CERs). In particular, the CERs have drawn much attention and several reported studies demonstrate that changes in CER composition may play a role in an impaired skin barrier ( 5-11 ). The various CERs consist of a sphingoid base linked via an amide bond to a fatty acid. Both variations in the fatty acid carbon chain and the sphingoid base architecture result in a large number of CER subclasses with a wide variation in chain length distribution. The molecular architecture of these subclasses is depicted in Fig. 1 . Over the years, important information about CER composition in human SC has been obtained using high performance thin layer chromatography (HPTLC) in conjunction with NMR. This resulted in a gradual increase in the number of identifi ed subclasses. By use of these methods, nine different human CER subclasses have been identifi ed in the SC of healthy human skin ( 12-16 ). As HPTLC is easily accessible, it is still frequently used in various fi elds with respect to skin lipid research to determine the CER subclasses in human SC ( 11,17,18 ). However, these approaches are very time consuming and a high quantity of material is required, which is not always available. The application of LC/MS results in a much more detailed profi le of each individual subclass using only small quantities of material (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Besides their important role in human skin, CERs are also key molecules with respect to cell signaling, growth, differentiation, and apoptosis ( 29-38 ). Also in these research areas, the introAbstract Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identifi ed. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identifi ed subclasses, a 3D multimass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked -hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identifi cation of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from fullthickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profi le. The outermost layer of mammalian skin, the stratum corneum (SC), consists of corneocytes embedd...

1-O-acylceramides are natural components of human and mouse epidermis

“…Epidermis is one of the richest sources for Cers and displays a high Cer structural diversity ( 8,9 ). Van Smeden et al ( 9 ) and Wertz and Downing ( 36 ) pointed out the presence of a very hydrophobic series of unknown esterifi ed chain Cers ( 17 ).…”

“…Van Smeden et al ( 9 ) and Wertz and Downing ( 36 ) pointed out the presence of a very hydrophobic series of unknown esterifi ed chain Cers ( 17 ). We investigated the involvement of LPLA 2 in epidermal 1-O-acylceramide synthesis by comparing 1-Oacylceramide levels from extracts of controls and LPLA 2 mutants.…”

The physical chemistry of the stratum corneum lipids

“…The ceramide molecules consist of fatty acids amide linked to sphingoid bases. Fifteen ceramide subclasses have been identified so far in the extracellular space of SC [28][29][30]. The ceramide molecules have different headgroup architectures, all of them considerably smaller and less polar than those of the lipids in a typical phospholipid membrane.…”

“…Both the ceramides and the fatty acids of the SC have unusually long hydrocarbon chains with a broad distribution of lengths [29,30,[35][36][37]. The variety of chain lengths and headgroup structures results in the existence of over 400 different ceramide species [29,30,38]. Most common for the ceramides are lengths of [24][25][26] [37], whereas most common for the fatty acids are lengths of 22-26 carbon atoms [39].…”

“…The different classes of ceramides molecules can participate in different inter-and intramolecular interactions with the other lipid molecules and thus make distinct contributions to the organization and the structural integrity of the lipid matrix [31][32][33][34][35]. Both the ceramides and the fatty acids of the SC have unusually long hydrocarbon chains with a broad distribution of lengths [29,30,[35][36][37]. The variety of chain lengths and headgroup structures results in the existence of over 400 different ceramide species [29,30,38].…”