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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, phytosph-ingosine- 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.

SUBMITTER: Kawana M

PROVIDER: S-EPMC7269764 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description: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 identified. 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 identified subclasses, a 3D multi-mass 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 ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification 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 full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.

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Comprehensive quantification of ceramide species in human stratum corneum.

Project description:One of the key challenges in lipidomics is to quantify lipidomes of interest, as it is practically impossible to collect all authentic materials covering the targeted lipidomes. For diverse ceramides (CER) in human stratum corneum (SC) that play important physicochemical roles in the skin, we developed a novel method for quantification of the overall CER species by improving our previously reported profiling technique using normal-phase liquid chromatography-electrospray ionization-mass spectrometry (NPLC-ESI-MS). The use of simultaneous selected ion monitoring measurement of as many as 182 kinds of molecular-related ions enables the highly sensitive detection of the overall CER species, as they can be analyzed in only one SC-stripped tape as small as 5 mm x 10 mm. To comprehensively quantify CERs, including those not available as authentic species, we designed a procedure to estimate their levels using relative responses of representative authentic species covering the species targeted, considering the systematic error based on intra-/inter-day analyses. The CER levels obtained by this method were comparable to those determined by conventional thin-layer chromatography (TLC), which guarantees the validity of this method. This method opens lipidomics approaches for CERs in the SC.

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Reduction of stratum corneum ceramides in Neu-Laxova syndrome caused by phosphoglycerate dehydrogenase deficiency.

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Topically Applied Ceramides Interact with the Stratum Corneum Lipid Matrix in Compromised Ex Vivo Skin.

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