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

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
DOI 10.1194/jlr.ra120000671PMC PMC7269764
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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.

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.

Claims the paper makes

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

  • Premisecitation

    The fragment ion pattern of ceramides differs depending on their long-chain base moiety.

    “Additionally, the fragment ion pattern of ceramides differs depending on their LCB moiety (5).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [5] Masukawa, 2008, Characterization of overall ceramide species in human stratum corneum, J. Lipid Res. source
  • Premisecitation

    The composition of ceramide classes/species in the human stratum corneum has previously been analyzed using LC/MS.

    “The composition of ceramide classes/species in the human SC has previously been analyzed using LC/MS (5-7, 16, 17, 29, 30).”
    Quote in the paperSubstantial claim3 of 7 sources checked
    • cites [5] Masukawa, 2008, Characterization of overall ceramide species in human stratum corneum, J. Lipid Res. source
    • cites [6] van Smeden, 2011, LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery, J. Lipid Res. source
    • cites [7] t'Kindt, 2012, Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry, Anal. Chem. source
    • cites [16] Ishikawa, 2010, Changes in the ceramide profile of atopic dermatitis patients, J. Invest. Dermatol. source
    • cites [17] Janssens, 2012, Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients, J. Lipid Res. source
    • cites [29] Farwanah, 2005, Profiling of human stratum corneum ceramides by means of normal phase LC/APCI-MS, Anal. Bioanal. Chem. source
    • cites [30] Weerheim, 2001, Determination of stratum corneum lipid profile by tape stripping in combination with high-performance thin-layer chromatography, Arch. Dermatol. Res. source
  • Premisecitation

    The fragmentation pattern of the peak at 12.86 min is consistent with that of C26:0 BS in previous reports.

    “The fragmentation pattern of this peak is consistent with that of C26:0 BS in previous reports (12,42), demonstrating that the peak detected at 12.86 min was C26:0 BS.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [12] Tsugawa, 2017, Comprehensive identification of sphingolipid species by in silico retention time and tandem mass spectral library, J. Cheminform. source
    • cites [42] Hsu, 2016, Complete structural characterization of ceramides as [M-H]– ions by multiple-stage linear ion trap mass spectrometry, Biochimie. source
  • Premisecitation

    More than several hundred ceramide species exist in the human stratum corneum.

    “Therefore, more than several hundred ceramide species exist in the human SC (5)(6)(7).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [5] Masukawa, 2008, Characterization of overall ceramide species in human stratum corneum, J. Lipid Res. source
    • cites [6] van Smeden, 2011, LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery, J. Lipid Res. source

      The Abstract reports 11 identified ceramide subclasses in human stratum corneum, and the Results and Discussion describe variation in their chain lengths. These passages support the existence of many distinct ceramides, but our copy does not establish that the number of species exceeds several hundred. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [7] t'Kindt, 2012, Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry, Anal. Chem. source
  • Interpretationcitation

    The peak detected at 12.86 min in mouse epidermis was C26:0 BS.

    “The fragmentation pattern of this peak is consistent with that of C26:0 BS in previous reports (12,42), demonstrating that the peak detected at 12.86 min was C26:0 BS.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [12] Tsugawa, 2017, Comprehensive identification of sphingolipid species by in silico retention time and tandem mass spectral library, J. Cheminform. source

      The Results and discussion section, under “Annotation of β-hydroxy ceramide,” identifies an experimental MS/MS spectrum as Ceramide [BS] (d18:1/26:0) and reports Ceramide [BS] in murine ear tissue. It does not identify a peak at 12.86 min or report that measurement in mouse epidermis, so those specifics are not supported by this source. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [42] Hsu, 2016, Complete structural characterization of ceramides as [M-H]– ions by multiple-stage linear ion trap mass spectrometry, Biochimie. source
  • Classified as probably not a substantial claim. 8
  • May combine two claims. 26

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In the human stratum corneum, 16 ceramide classes other than SD and β-hydroxy ceramides have been identified.

    “In the human SC, 16 ceramide classes other than SD and -hydroxy ceramides have been identified (5)(6)(7).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [5] Masukawa, 2008, Characterization of overall ceramide species in human stratum corneum, J. Lipid Res. source

      The Nomenclature section describes 12 ceramide classes, including classes not previously reported, rather than 16 excluding SD and β-hydroxy ceramides. The Discussion reports identifying species within 11 classes; our copy does not substantiate the claimed count of 16. (LLM, conf=0.87)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [6] van Smeden, 2011, LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery, J. Lipid Res. source

      The Abstract reports 11 previously identified ceramide subclasses in human stratum corneum and identifies one additional subclass, CER [EOdS]. The Results and Discussion likewise describe 12 subclasses as the theoretical total, contrary to the attributed count of 16 even before the stated exclusions. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [7] t'Kindt, 2012, Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry, Anal. Chem. source

  • May combine two claims.

    Premisecitation

    In Fa2h KO mice, neither the skin phenotype nor reduced AS levels in skin were observed.

    “In Fa2h KO mice, neither the skin phenotype nor reduced AS levels in skin were observed (53).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [53] Maier, 2011, Normal fur development and sebum production depends on fatty acid 2-hydroxylase expression in sebaceous glands, J. Biol. Chem. source

      In “Delayed Fur Emergence and Cyclic Alopecia in FA2H-deficient Mice” (page unavailable), the paper reports delayed fur development and repeated hair loss in Fa2h knockout mice—a skin phenotype, contrary to the claim. Its lipid analysis also reports reduced hydroxylated glucosylceramides in total skin, although hydroxylated ceramides showed no obvious change. The copy does not identify these measurements as “AS levels.” (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    A portion of EO ceramides are converted into protein-bound ω-hydroxy ceramides, which are connected to cross-linked proteins constituting the cornified envelope.

    “A portion of EO ceramides are converted into protein-bound hydroxy (P-O) ceramides (Fig. 1B), which are connected to cross-linked proteins constituting the cornified envelope, a structure located beneath the surface of corneocytes (20)(21)(22).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [20] Candi, 2005, The cornified envelope: a model of cell death in the skin, Nat. Rev. Mol. Cell Biol. source
    • cites [21] Elias, 2014, Formation and functions of the corneocyte lipid envelope (CLE), Biochim. Biophys. Acta. source
    • cites [22] Akiyama, 2017, Corneocyte lipid envelope (CLE), the key structure for skin barrier function and ichthyosis pathogenesis, J. Dermatol. Sci. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    EO ceramides are epidermis-specific ceramide classes.

    “EO ceramides are epidermisspecific ceramide classes, and their characteristic EO structure is important for the formation and stabilization of lipid organization in the lipid lamellae (18,19).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [18] Bouwstra, 2002, Phase behavior of stratum corneum lipid mixtures based on human ceramides: the role of natural and synthetic ceramide 1, J. Invest. Dermatol. source

      The paper’s introductory discussion describes human ceramides 1 and 4 with the characteristic ester-linked structure as ceramides isolated from the stratum corneum. It does not establish that these classes are specific to the epidermis rather than also occurring elsewhere. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [19] Lundborg, 2018, Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation, J. Struct. Biol. source

      Section 4.2 (page unavailable) identifies CER EOS as a component of the human stratum corneum lipid structure, and section 4.1 reports that adding it brings the model’s lamellar periodicity closer to the observed value. The copy does not establish that EO ceramide classes are specific to the epidermis, nor does it directly show that their EO structure stabilizes the lamellae. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The characteristic EO structure is important for the formation and stabilization of lipid organization in the lipid lamellae.

    “EO ceramides are epidermisspecific ceramide classes, and their characteristic EO structure is important for the formation and stabilization of lipid organization in the lipid lamellae (18,19).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [18] Bouwstra, 2002, Phase behavior of stratum corneum lipid mixtures based on human ceramides: the role of natural and synthetic ceramide 1, J. Invest. Dermatol. source

      The Discussion (page unavailable) concludes that human ceramide 1 plays an important role in forming the long-periodicity lamellar phase and that its acyl-chain structure affects lamellar organization. This supports the formation part of the claim, but the paper does not establish that the EO structure stabilizes an existing lipid organization. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [19] Lundborg, 2018, Human skin barrier structure and function analyzed by cryo-EM and molecular dynamics simulation, J. Struct. Biol. source

      In Results §3.1 (page not available), changing CER EOS content alters the modeled lipid lamellae’s periodicity, and a model containing 5% CER EOS best matches the cryo-EM images. Discussion §4.1 likewise says adding CER EOS helps reproduce the observed periodicity. These findings support a role in lamellar organization, but the paper does not establish that the characteristic EO structure is important for its formation or stabilization. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    In that report, it is likely that BS, but not AS, was measured, because AS levels were determined by TLC or MS analysis without LC separation.

    “In that report, however, it is likely that BS, but not AS, was measured, because AS levels were determined by TLC or MS analysis without LC separation (53).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [53] Maier, 2011, Normal fur development and sebum production depends on fatty acid 2-hydroxylase expression in sebaceous glands, J. Biol. Chem. source

      The Experimental Procedures (page not available) describe TLC analysis and direct injection into the mass spectrometer, supporting the claim that no LC separation preceded MS. The paper groups shorter-chain hydroxylated sphingolipids as α-hydroxy based on chain length, but does not establish that the measured material was likely BS rather than AS. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Earlier studies have reported that ceramides with odd-chain FAs exist at high levels in the SC.

    “Earlier studies have reported that ceramides with oddchain FAs exist at high levels in the SC (Fig. 3F, supplemental Table S3) (7,55).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [7] t'Kindt, 2012, Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry, Anal. Chem. source
    • cites [55] Masukawa, 2009, Comprehensive quantification of ceramide species in human stratum corneum, J. Lipid Res. source

      The Methods passage lists an odd-chain ceramide, CER[N(17)S(18)], as an internal standard, not as a ceramide found at high levels in stratum corneum. Searches for odd-chain fatty acids and their abundance found no passage in our copy reporting the claimed high levels; the paper discusses quantifying ceramide species generally. (LLM, conf=0.72)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Sphingosine- and phytosphingosine-type ceramides are generated either via desaturation between the 4 and 5 positions of the LCB moiety of dihydroceramides (DS ceramides) by DEGS1, or via hydroxylation of the same moiety at the 4 position by DEGS2.

    “Sphingosine-and phytosphingosine-type ceramides are generated either via desaturation between the 4 and 5 positions of the LCB moiety of dihydroceramides (DS ceramides) by the desaturase DEGS1, or via hydroxylation of the same moiety at the 4 position by the hydroxylase DEGS2 (49,50).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [49] Omae, 2004, DES2 protein is responsible for phytoceramide biosynthesis in the mouse small intestine, Biochem. J. source
    • cites [50] Ternes, 2002, Identification and characterization of a sphingolipid Δ4-desaturase family, J. Biol. Chem. source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    LCBs with different chain lengths (C14-C26) reportedly exist in the epidermis.

    “Although we focused on and quantified ceramide species containing LCBs with a C18 chain length, LCBs with different chain lengths (C14-C26) reportedly exist in the epidermis (57).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [57] Pons, 2002, Gas-chromatography/mass-spectrometry analysis of human skin constituents as heptafluorobutyrate derivatives with special reference to long-chain bases, J. Lipid Res. source

      Table 2 (shown under “Conclusion and Perspectives”; page unavailable) identifies LCBs with multiple chain lengths, including C16 through C26, in a human skin extract. It does not identify a C14 LCB, and the analysis is of skin extracts rather than the epidermis specifically. The claimed C14–C26 range in the epidermis is therefore only partly supported. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Dehydrated AS was detected predominantly, whereas nondehydrated OS was detected at the same levels as dehydrated OS.

    “Dehydrated AS was also detected predominantly, whereas nondehydrated OS was detected at the same levels as dehydrated OS.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In LC/MS/MS analysis using an MRM setting to detect C26:0 AS, C26:0 AS was detected in human stratum corneum at 13.20 min, while a large peak at 12.86 min was detected in mouse epidermis.

    “In LC/MS/MS analysis using the MRM setting to detect C26:0 AS, C26:0 AS was detected in the human SC (13.20 min), while a large peak at an earlier retention time (12.86 min), instead of C26:0 AS, was detected in the mouse epidermis (Fig. 2D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Product ions of m/z 266 and 284 were mainly produced by fragmentation of NDS, and neither were detected in other ceramide classes.

    “Product ions of m/z 266 and 284 were mainly produced by the fragmentation of NDS, and neither were detected in other ceramide classes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Ceramide classes with double bond(s) in their long-chain base (NS, NH, and NSD) were highly dehydrated, while those without double bonds (NDS and NP) were not.

    “Ceramide classes with double bond(s) in their LCB (NS, NH, and NSD) were highly dehydrated, while those without double bonds (NDS and NP) were not (Fig. 2F).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The fatty acid compositions of human AH, human AP, and mouse BS were C26:0 > C24:0 > C25:0; C24:0 > C26:0 > C25:0; and C26:0 > C28:0 > C25:0, respectively.

    “The FA compositions of the human AH, AP, and mouse BS were C26:0 > C24:0 > C25:0; C24:0 > C26:0 > C25:0; and C26:0 > C28:0 > C25:0, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Generally, the fatty acid chain length of ω-hydroxylated ceramides (EO and ω-hydroxy) was C30-C34, and that of other ceramides was C16-C30.

    “Generally, the FA chain length of -hydroxylated ceramides (EO and -hydroxy) was C30-C34, and that of the other ceramides was C16-C30.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In human SC P-O ceramides, P-OS was the most abundant, and P-OH and P-OP were half and 1/10 of P-OS, respectively.

    “The amounts of EO ceramides were comparable between EOS, EOH, and EOP in the human SC (Fig. 3A), whereas those of P-O ceramides were different: P-OS was the most abundant, and P-OH and P-OP were half and 1/10 of P-OS, respectively (Fig. 4A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    C26:0 and C24:0 were the top two fatty acids in most ceramide classes, other than ω-hydroxy or EO ceramides, although C16:0 was the principal fatty acid in human AS.

    “C26:0 and C24:0 were the top two FAs in most ceramide classes, other than -hydroxy or EO ceramides, although C16:0 was the principal ceramide present in human AS (C16:0 > C26:0 > C24:0).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Major fatty acids of nonhydroxy ceramides were C26:0 > C24:0 > C28:0 both in human and mouse stratum corneum, except for human NDS, which was C24:0 > C26:0 > C28:0.

    “Major FAs of nonhydroxy ceramides were C26:0 > C24:0 > C28:0 both in human and mouse SC (except for human NDS, which was C24:0 > C26:0 > C28:0).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    FA species constituting P-OS are C30-C34, with C30:0 > C32:0 > C32:1 in humans and C34:1 ≈ C32:0 > C32:1 in mice.

    “FA species that constituted P-OS were C30-C34 (C30:0 > C32:0 > C32:1 in humans and C34:1 ≈ C32:0 > C32:1 in mice) (Fig. 4B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the LC chromatogram of synthesized (R,S) C18:0 BS, two peaks were observed at 8.63 and 8.78 min, and the latter peak was identical with C18:0 BS in mouse epidermis.

    “In the LC chromatogram of the synthesized (R, S) C18:0 BS, two peaks were observed at 8.63 and 8.78 min, and the latter peak was identical with C18:0 BS in the mouse epidermis (Fig. 5A).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    The ratio of each ceramide class to total ceramides in this study was not largely different from earlier studies.

    “The ratio of each ceramide class to total ceramides in this study was not largely different from earlier studies (supplemental Table S6).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In mouse SC, 16 free ceramide classes/268 species and 5 P-O ceramide classes/74 species were quantified.

    “In the current study, we performed LC/MS/MS analyses and quantified free ceramides (21 classes/345 species in humans; 16 classes/268 species in mice) and P-O ceramides (5 classes/63 species in humans; 5 classes/74 species in mice) (Figs. 3, 4; supplemental Tables S2-S5), revealing the in-depth ceramide profiles of human and mouse SC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    We successfully quantified 63 P-O ceramide species in humans and 74 P-O ceramide species in mice.

    “Although semiquantitative analysis of P-O ceramides by TLC and quantification of limited P-O ceramide species by LC/MS/MS have already been conducted (44-46), we successfully quantified 63 and 74 P-O ceramide species in humans and mice, respectively, leading to the elucidation of the detailed composition of P-O ceramides.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the LC chromatogram of (R) C14:0 BS, two peaks (6.79 and 6.96 min) were observed, and the latter peak was larger than the former.

    “Although two peaks (6.79 and 6.96 min) were also observed in (R) C14:0 BS, the latter peak was larger than the former, indicating that the former and latter peaks corresponded to (S) BS and (R) BS, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In human SC, 21 free ceramide classes/345 species and 5 P-O ceramide classes/63 species were quantified.

    “In the current study, we performed LC/MS/MS analyses and quantified free ceramides (21 classes/345 species in humans; 16 classes/268 species in mice) and P-O ceramides (5 classes/63 species in humans; 5 classes/74 species in mice) (Figs. 3, 4; supplemental Tables S2-S5), revealing the in-depth ceramide profiles of human and mouse SC.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mouse SC, phytosphingosine-type ceramides were at very low levels and 6-hydroxy sphingosine-type ceramides were undetectable.

    “On the other hand, phytosphingosine-and 6-hydroxy sphingosine-type ceramides were at very low and undetectable levels, respectively, in the mouse SC (Fig. 3; supplemental Tables S2, S3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    In humans, P-OS is the predominant P-O ceramide class, comprising 64% of total P-O ceramides, followed by P-OH at 28%.

    “Consistent with earlier reports (43,44), in humans, P-OS was predominant (64% of total P-O ceramides), followed by P-OH (28%) (Fig. 4A; supplemental Tables S4, S5).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [43] Robson, 1994, 6-Hydroxy-4-sphingenine in human epidermal ceramides, J. Lipid Res. source
    • cites [44] Wertz, 1989, Covalently bound lipids of human stratum corneum, J. Invest. Dermatol. source

  • May combine two claims.

    Findingown data

    Mouse EOS and OS contained C32:0 > C30:0 > C34:1 and C32:0 > C34:1 > C30:0, respectively.

    “Mouse EOS and OS contained C32:0 > C30:0 > C34:1 and C32:0 > C34:1 > C30:0, respectively.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Ceramides account for approximately 50% by weight of the main constituents of lipid lamellae in the stratum corneum.

    “The main constituents of lipid lamellae are ceramides, cholesterol, and FFAs, of which ceramides account for 50% by weight (4).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [4] Yardley, 1981, Lipid composition and metabolism in normal and diseased epidermis, Pharmacol. Ther. source

  • May combine two claims.

    Interpretationown data

    Although a matrix effect (suppression of ionization) is caused by tape-derived coextracts, this effect can be nullified by reducing the tape size to 50 mm2 or less.

    “From this result, although a matrix effect (suppression of ionization) is caused by the tape-derived coextracts, this effect can be nullified by reducing the tape size to 50 mm 2 or less.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    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 low activities against EOP and EOH of enzymes involved in P-O ceramide production.

    “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.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: P-O ceramides are derived from EO ceramides.

  • May combine two claims.

    Interpretationreasoning

    The absence of 6-hydroxy sphingosine-type ceramide in mice suggests that the gene involved does not exist in mice or is not expressed in the skin of mice.

    “Although the gene responsible for the production of 6-hydroxy sphingosine-type ceramide has not yet been identified, the absence of 6-hydroxy sphingosine-type ceramide in mice suggests that the gene involved does not exist in mice or is not expressed in the skin of mice.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The former and latter peaks in (R) C14:0 BS correspond to (S) BS and (R) BS, respectively.

    “Although two peaks (6.79 and 6.96 min) were also observed in (R) C14:0 BS, the latter peak was larger than the former, indicating that the former and latter peaks corresponded to (S) BS and (R) BS, respectively.”
    Quote in the paperSubstantial claim
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

Cites

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Authors
Kawana, M., Miyamoto, M., Ohno, Y., & Kihara, A.
Affiliations
Hokkaido University (JP)
Corresponding author
  • Ohno, Yusuke
  • Kihara, Akio
ORCID
Venue
Journal of Lipid Research
Publisher
Elsevier BV
Published
2020-06
Volume
61
Issue
6
Pages
884-895
ISSN
0022-2275 (ISSN-L)
DOI
10.1194/jlr.ra120000671
PMC
PMC7269764
OpenAlex
W3015053316
Semantic Scholar
d636fe0987fbf6e6a53d593f9c3e15494bebdae9
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
Licence
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

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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