Cited by 129Sphingolipid Metabolism and SignalingAdvancements in Transdermal Drug DeliveryDermatology and Skin Diseases

The DOI matches its Crossref record; 59 of the 59 references it cites were found in a registry or an index; 25 of the 39 sources its claims cite were checked for support.

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

Open access
Kawana, Momoko, Miyamoto, Masatoshi, Ohno, Yusuke, Kihara, Akio·2020·Journal of Lipid Research

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.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

The paper establishes an LC/MS/MS method using deuterated standards to quantify free and protein-bound ceramides in human and mouse stratum corneum, revealing large class and species differences. Humans are rich in phytosphingosine- and 6-hydroxy sphingosine-type ceramides and α-hydroxy ceramides, whereas mice are dominated by sphingosine-type ceramides and contain more β-/ω-hydroxy and EO ceramides.

Key findings

  • The method quantified 21 free ceramide classes/345 species in human SC and 16 classes/268 species in mouse SC; P-O ceramides were quantified as 5 classes/63 species in humans and 5 classes/74 species in mice.
  • In human SC, NP, NH, and AH were abundant (24.2%, 23.7%, and 18.0% of total ceramides, respectively); 6-hydroxy sphingosine- and phytosphingosine-type ceramides accounted for 45.4% and 34.7%. In mouse SC, NS was highest (57.9%), sphingosine-type ceramides accounted for 87.7%, phytosphingosine-type ceramides were 1.2%, and 6-hydroxy sphingosine-type ceramides were undetectable.
  • By FA type, human SC was 59.4% nonhydroxy, 32.5% α-hydroxy, 6.3% EO, 1.6% ω-hydroxy, and 0.17% β-hydroxy ceramides; mouse SC was 68.9% nonhydroxy, 16.9% EO, 8.7% ω-hydroxy, 4.9% β-hydroxy, and 0.7% α-hydroxy ceramides.
  • The hydroxylated β-carbon in BS was in the (R) configuration, and HACD1/HACD2 double KO HAP1 cells showed a 15-fold increase in BS, suggesting β-hydroxy acyl-CoA from the ER FA elongation cycle is a substrate for BS synthesis.

Methods

  • Human SC was collected by tape stripping from healthy volunteers (10 males and 9 females aged 20–50 y; n = 19); mouse SC was collected from the back of C57BL/6J mice on the first day after birth (n = 3), using the second tape strip for lipid extraction.
  • Ceramides were analyzed by UPLC coupled to ESI tandem quadrupole MS in MRM mode with a reverse-phase C18 column and deuterium-labeled ceramide standards; matrix effects from tape extracts were tested, and P-O ceramides were released by KOH/methanol extraction.
  • In vitro assays used HEK293T cells overexpressing CERS1 or CERS5 for BS synthesis and HACD1/HACD2 double KO HAP1 cells to test BS levels.

Limitations

  • The paper states that it focused on and quantified ceramide species containing C18 chain-length LCBs, while LCBs with different chain lengths (C14–C26) reportedly exist in the epidermis and remain for future analysis.
  • The paper states that further analyses are required for BS synthesis because (R)-β-hydroxy acyl-CoAs are also produced in peroxisomal β-oxidation and BS could alternatively be generated by β-hydroxylation of NS by an unidentified hydroxylase.
  • The paper notes that mice deficient in a hydroxylase gene involved in hydroxylated ceramide production may not always be appropriate models for human skin disorders; evident from the text, the mouse sample was small (n = 3), from a single early time point and back skin, and no functional barrier measurements are reported.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1194/jlr.ra120000671

Cites

59 of 59 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

25 of 39 cited sources checked · 10 supported · 3 flagged · accuracy not yet measured

Show all 61 claims

  • Probably not a substantial claim. 8
  • It may state two separate claims as one. 26
33 of 33
  • The paper relies on this without support.

    The appropriate maintenance of the amount and composition of stratum corneum ceramides is important for normal skin barrier function.

    “The appropriate maintenance of the amount and composition of SC ceramides is important for normal skin barrier function.”
  • The paper relies on this without support.

    It is currently unclear whether 4E,14Z-sphingadiene is present in the epidermis.

    “It is currently unclear whether SD is present in the epidermis.”
  • The paper relies on this without support.

    The presence of β-hydroxy ceramides in human skin has not yet been determined.

    “Although ceramides containing -hydroxy FA have recently been identified in the mouse epidermis (11,12), their presence in human skin has not yet been determined.”
  • The paper relies on this without support.

    The degree of dehydration of each ceramide class has not yet been determined.

    “However, the degree of dehydration of each ceramide class has not yet been determined.”
  • The paper relies on this without support.

    The amounts of ceramide classes other than sphingosine-type ceramides in mice have not yet been elucidated.

    “Thus, although mice have been generally utilized as a model animal for studying skin barrier function, the ceramide classes measured have primarily been sphingosine-type ceramides such as NS and EOS, and the amounts of other ceramide classes have not yet been elucidated.”
  • The paper relies on this without support.

    The matrix effect caused by coextracted compounds from the tape is unknown.

    “Although an SC sample is often collected by tape stripping, the matrix effect caused by coextracted compounds from the tape is unknown.”
  • The paper relies on this without support.

    It is difficult to separately detect and precisely quantify a tremendous number of ceramide species by LC/MS.

    “However, it is difficult to separately detect and precisely quantify such a tremendous number of ceramide species by LC/MS.”
  • The paper relies on this without support.

    P-O ceramides are derived from EO ceramides.

    “Because P-O ceramides are derived from EO ceramides, the difference in LCB composition between EO and P-O ceramides indicates that the conversion efficiency of EO to P-O ceramides varies depending on the LCB structure (the presence of hydroxylation), presumably due to the low activities against EOP and EOH of the enzymes involved in the production of P-O ceramides.”
  • The paper relies on this without support.

    The amounts of SD-type ceramides and BS were previously unknown.

    “Although the amounts of SDtype ceramides and BS were previously unknown, we determined them for the first time (Fig. 3; supplemental Tables S2, S3).”
9 of 9

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • NSD synthesis incubation time differs between Methods and Fig. 2

    Minor

    The Methods specify a 30-minute incubation for NSD synthesis, while the Fig. 2 caption specifies 1 hour for the same synthesis.

1 of 1

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Metadata

Authors
Kawana, M., Miyamoto, M., Ohno, Y., & Kihara, A.
Affiliations
Hokkaido University (JP)
Corresponding author
  • Ohno, Yusuke
  • Kihara, Akio
Venue
Journal of Lipid Research
Publisher
Elsevier BV
Published
2020-06
Volume
61
Issue
6
Pages
884-895
ISSN
0022-2275 (ISSN-L)
OpenAlex
W3015053316
Cited by
129
References
57
Citations by year
2026: 17 · 2025: 20 · 2024: 31 · 2023: 23 · 2022: 17 · 2021: 19 · 2020: 2
FWCI
4.21
Type
journal article
Language
en
Open access
Locations
  • Journal of Lipid Research (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Institutional Repositories DataBase (IRDB) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Hokkaido University Collection of Scholarly and Academic Papers (Hokkaido University) (submittedVersion, cc-by)
Funders
  • Japan Agency for Medical Research and Development (JP18H03976, JP15H05589)
  • Japan Society for the Promotion of Science
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sphingolipid Metabolism and Signaling
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Advancements in Transdermal Drug Delivery
  • Health Sciences › Medicine › Dermatology › Dermatology and Skin Diseases
Keywords
ceramide, stratum corneum, skin barrier function, human-mouse comparison, phytosphingosine, fatty acid elongation, HAP1 cells
MeSH terms
Lipidomics, Animals, Ceramides, Chromatography, Liquid, Epidermis, Humans, Mice, Tandem Mass Spectrometry

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