Ceramides From Epidermis

As previously noted, the first comprehensive study of epidermal ceramide structures was directed at the porcine ceramides, which were separated into six chromatographically distinct fractions [3]. Each fraction was analyzed by a combination of chemical, chromatographic and spectroscopic methods, and representative structures are included in Figure 1.

The least polar of the porcine ceramides, ceramide fraction 1, consists of 30- through 34-carbon m-hydroxyacids amide-linked to a mixture of sphingosines and dihydrosphingo-sines. The long-chain base component of this ceramide ranges from 16 through 22 carbons in length with 18:1, 20:1, and 22:1 being the most abundant. There is also a fatty acid ester-linked to the m-hydroxyl group, 75% of which consists of linoleic acid. This species has often been referred to as ceramide 1 or acylceramide, but in the more systematic nomenclature system proposed by Motta et al. [18] this becomes Cer[OSE]. (In this system, the amide-linked fatty acid is designated as N, A, or O to indicate normal, a-hydroxy, or m-hydroxy, respectively. The base component is designated S or P for sphingosine or

Figure 1 Representative structures of the free ceramides from human stratum corneum.

phytosphingosine, respectively. It is understood that sphingosines are generally accompanied by dihydrosphingosines in the ceramides.) Cer[OSE] is unusual in two respects: (1) the very long m-hydroxyacyl portion of the molecule is long enough to completely span a typical bilayer; and (2) a high proportion of the ester-linked fatty acid is linoleic acid. It is thought that this ceramide along with an analogous glucosylated Cer[OSE] in the living layers of the epidermis account for the essential role of linoleic acid in formation and maintenance of the barrier function of the skin [3,19,20]. Specific roles for Cer[OSE] have been proposed in organization of the intercellular lipid lamellae of epidermal stratum corneum [20-22]. In formation of the intercellular lamellae of the stratum corneum, flattened lipid vesicles are initially extruded from the lamellar granules into the intercellular space [23]. These flattened vesicles fuse in an edge-to-edge manner to produce paired bilayers. Cer[OSE] is associated with each of the paired lamellae with both possible orientations.

Approximately half of the Cer[OSE] is oriented with the polar head groups in the outer polar regions of the paired bilayers, whereas the other half of the Cer[OSE] molecules are oriented with the polar head groups in the polar regions in the center of the pair of lamellae. For the Cer[OSE] in the former orientation the ra-hydroxyacyl portion of the

molecule will span the bilayer while the linoleate inserts into the other bilayer, thus linking the pair of bilayers together. For Cer[OSE] in the second orientation the linoleate tail is thought to participate in the formation of narrow interdigitated layers that intervene between the paired bilayers. This action of the Cer[OSE] results in the formation of broad-narrow-broad lamellar patterns that are seen in transmission electron micrographs when ruthenium tetroxide is used as a postfixative and which give rise to a 13 nm repeat unit in radiograph diffraction studies [5,6,22].

Porcine ceramide fraction 2 has proven to be Cer[NS]. The fatty acid component is saturated and straight-chained and ranges from 16- through 32-carbons in length. C20:0, C22:0, C24:0, C26:0, and C28:0 are the most abundant, constituting from 9% to 19% of the total fatty acid mass each. The long-chain bases again consist of a mixture of sphingosines and dihydrosphingosines ranging from 16- through 22-carbons in length. The most abundant bases are 18:0, 18:1, 20:0, and 20:1.

Porcine ceramide fraction 3, Cer[NP], contains the same range of fatty acids found in Cer[NS], but the long-chain base component is now a phytosphingosine with no double bond and a third hydroxyl group on carbon 4. The phytosphingosines found here range from 16- through 24-carbons long, and the most abundant are 20:0 and 22:0.

Porcine ceramide fractions 4 and 5 both proved to be Cer[AS], but they differed in terms of the chain length distributions of the a-hydroxyacid component. The chromato-graphically more mobile fraction 4 contained 24- through 28-carbon a-hydroxyacids amide-linked to sphingosines and dihydrosphingosines, whereas ceramide fraction 5 contains a-hydroxypalmitic acid amide-linked to sphingosines and dihydrosphingosines. Ceramide fraction 4 also contains somewhat longer bases with major amounts of 20:0 and 20:1, whereas ceramide fraction 5 contains mainly 16- through 18-carbon bases. This difference in carbon content results in chromatographic separation into two fractions, even though the basic structural type is the same in each.

Finally, the most polar of the pig ceramide fractions consists of a-hydroxyacids amide-linked to phytosphingosine, Cer[AP]. The a-hydroxyacids present in Cer[AP] range from 16- through 28-carbons in length, but the 24- and 26-carbon entities account for approximately 70% of the total fatty acid mass. The phytosphingosines have a chain-length distribution similar to that already described for Cer[NP].

Subsequently, the human stratum corneum ceramides were investigated and were shown to produce a similar, though not identical, pattern on thin-layer chromatograms [15]. Notably, the human fraction most closely matching porcine ceramide fraction 3 is somewhat broader and less symmetrical. The material most closely matching porcine cera-mide fractions 4 and 5 merged into one broad peak, and was designated ceramide 4/5. This was shown to reflect a more continuous chain-length distribution among the a-hydroxyacid component of Cer[AS] as opposed to the bipolar distribution found in the pig. The most polar human fraction similar to porcine ceramide fraction 6 appeared as an incompletely resolved doublet. These two components were designated ceramides 6I and 6II. Subsequently it has been shown the ceramide fraction 6II contains the variant phytosphingosine—6-hydroxysphingosine [16]. The Motta system of nomenclature has been extended to include this new long-chain base as H [16]. So ceramide 6I is Cer[AP], and ceramide 6II becomes Cer[AH]. Human ceramide fraction 3 has been shown to contain a minor amount of a 6-hydroxysphingosine-containing acylceramide, Cer[OHE] [16], in addition to Cer[NH]. Likewise, ceramide fraction 4/5 contains Cer[NH] [24] in addition to Cer[AS] [15]. These additional ceramides containing 6-hydroxysphingosine can be resolved on thin-layer chromatography by use of multiple development regimens.

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Figure 2 Representative structures of the covalently bound ceramides from human stratum corneum.

In addition to the extractable lipids, there are covalently bound lipids coating the outer surface of the cornified envelope in epidermal stratum corneum. This consists mainly of ceramides. In porcine stratum corneum the principal covalently bound lipid is Cer[OS] derived from Cer[OSE] [4]. In human stratum corneum, in addition to covalently bound Cer[OS], a second more polar covalently bound ceramide was found [17]. This was later shown to be Cer[OH] [16]. Representative structures of Cer[OS] and Cer[OH] are presented in Figure 2.

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