Comparative profiling and comprehensive quantification of stratum corneum ceramides in humans and mice by LC/MS/MS

Abstract

Ceramides are the predominant lipids in the stratum corneum (SC) and are crucial components for normal skin barrier function. Although the composition of various ceramide classes in the human SC has been reported, that in mice is still unknown, despite mice being widely used as animal models of skin barrier function. Here, we performed LC/MS/MS analyses using recently available ceramide class standards to measure 25 classes of free ceramides and 5 classes of protein-bound ceramides from human and mouse SC. Phytosphingosine- and 6-hydroxy sphingosine-type ceramides, which both contain an additional hydroxyl group, were abundant in the human SC (35% and 45% of total ceramides, respectively). In contrast, in mice, phytosph­ingosine- and 6-hydroxy sphingosine-type ceramides were present at ∼1% and undetectable levels, respectively, and sphingosine-type ceramides accounted for ∼90%. In humans, ceramides containing α-hydroxy FA were abundant, whereas ceramides containing β-hydroxy or ω-hydroxy FA were abundant in mice. The hydroxylated β-carbon in β-hydroxy ceramides was in the (R) configuration. Genetic knockout of β-hydroxy acyl-CoA dehydratases in HAP1 cells increased β-hydroxy ceramide levels, suggesting that β-hydroxy acyl-CoA, an FA-elongation cycle intermediate in the ER, is a substrate for β-hydroxy ceramide synthesis. We anticipate that our methods and findings will help to elucidate the role of each ceramide class in skin barrier formation and in the pathogenesis of skin disorders.

Dietary ceramide 2-aminoethylphosphonate, a marine sphingophosphonolipid, improves skin barrier function in hairless mice

“…In this study, administration of CAEP and GluCer increased epidermal covalently bound ω-hydroxyceramide levels and its synthesis-related gene expression in the skin compared to the control group. We found major covalently bound ω-hydroxyceramides in mice, although the minor molecule species (dihydrosphingosine-type, 4,14-sphingadiene-type and sphingosine-type involving odd-numbered carbon chain fatty acid) indicated by Kawana et al 62 were not detected. Dietary CAEP and GluCer intake effectively restore the skin condition in this mouse model, because ingestion of CAEP and GluCer normalizes the levels of epidermal ω-hydroxyceramide in mice compared to normal, hairless mice (data not shown).…”

Comprehensive stratum corneum ceramide profiling reveals reduced acylceramides in ichthyosis patient with CERS3 mutations

“…The organic phase was recovered and dried. Lipids were dissolved in chloroform/methanol (1:2, v/v), and d 7 -C26:0 NS ceramide was analyzed using LC-MS/MS as described previously, 9,20…”

“…SC samples from the skin of the back were collected from the male ichthyosis patient (aged 1 year) and three healthy male individuals (aged 1, 2, and 31 years), as described previously. 9 From each tape strip, three 10 mm × 10 mm pieces were cut, which were used as replicates of that sample. The extraction of lipids from tape strips, preparation of free and protein-bound ceramides, and LC-MS/MS quantification of these species as well as the internal standard lipids used were performed as described previously.…”

“…The extraction of lipids from tape strips, preparation of free and protein-bound ceramides, and LC-MS/MS quantification of these species as well as the internal standard lipids used were performed as described previously. 9 Briefly, methanol (800 μl) was added to the tape to extract the lipids. After the tape was removed, the samples were centrifuged.…”

“…1b). 6,9 In addition, one of the three FA types (non-hydroxy, α-hydroxy, and ω-hydroxy) is present in human ceramides; these differ in the number and position of the hydroxyl group (Fig. 1c).…”