Skip to main content
SpringerLink
Log in

Comprehensive characterization and simultaneous analysis of overall lipids in reconstructed human epidermis using NPLC/HR-MSn: 1-O-E (EO) Cer, a new ceramide subclass

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Stratum corneum lipids are responsible for the skin’s barrier function. They are the final product of epidermis lipid biosynthesis. During this process, lipids evolve from simple to complex structures in three main levels respectively (stratum basal level, stratum granulosum level, and stratum corneum level). Our aim was to simultaneously analyze and characterize the structure of total epidermis lipids. A powerful analytical method (normal-phase liquid chromatography coupled with high-resolution mass spectrometry (NPLC/HR-MSn)) was developed in order to separate, in a single run, lipid classes with a wide polarity range. Chromatographic conditions were particularly designed to analyze lipids of intermediate polarity such as ceramides. Rich information was obtained about the molecular structure of keratinocyte differentiation biomarkers such as ceramides, glucosylceramides, and sphingomyelins and the microstructures of reconstructed human epidermis lipids using HR-MSn. A new subclass of ceramides, 1-O-Acyl Omega-linoleoyloxy ceramides [1-O-E (EO) Cer] has been highlighted. This class is double esterified on the 1-O-position of sphingoid base with long to very long chain acyl residues (1-O-E) and on the position of ω-hydroxyl group of fatty acid with the linolenic acid (EO). Considering its chemical structure and hydrophobicity, this subclass can contribute to the skin barrier. In addition, we detected a new epidermis sphingomyelins. Our lipidomic approach offers a direct access to epidermis biomarkers.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

APCI:

Atmospheric pressure chemical ionization

APPI:

Atmospheric pressure photoionization

CER:

Ceramide

ESI:

Electrospray ionization

FFA:

Free fatty acid

GlcCER:

Glucosylceramide

HPLC:

High-performance liquid chromatography

HR-MS:

High-resolution mass spectrometry

LA:

Linoleic acid

NPLC:

Normal-phase liquid chromatography

RHE:

Reconstructed human epidermis

RP-LC:

Reversed-phase liquid chromatography

SC:

Stratum corneum

SM:

Sphingomyelin

Sph:

Sphingoid base moiety

ULC:

Ultra-long chain

References

  1. Breiden B, Gallala H, Doering T, Sandhoff K. Optimization of submerged keratinocyte cultures for the synthesis of barrier ceramides. Eur J Cell Biol. 2007;86(11–12):657–73.

    CAS  PubMed  Google Scholar 

  2. Franzke CW, Cobzaru C, Triantafyllopoulou A, Loffek S, Horiuchi K, Threadgill DW, et al. Epidermal ADAM17 maintains the skin barrier by regulating EGFR ligand-dependent terminal keratinocyte differentiation. J Exp Med. 2012;209(6):1105–19.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. van Drongelen V, Alloul-Ramdhani M, Danso MO, Mieremet A, Mulder A, van Smeden J, et al. Knock-down of filaggrin does not affect lipid organization and composition in stratum corneum of reconstructed human skin equivalents. Exp Dermatol. 2013;22(12):807–12.

    PubMed  Google Scholar 

  4. Vyumvuhore R, Tfayli A, Duplan H, Delalleau A, Manfait M, Baillet-Guffroy A. Effects of atmospheric relative humidity on stratum corneum structure at the molecular level: ex vivo Raman spectroscopy analysis. Analyst. 2013;138(14):4103–11.

    CAS  PubMed  Google Scholar 

  5. Sahle FF, Gebre-Mariam T, Dobner B, Wohlrab J, Neubert RH. Skin diseases associated with the depletion of stratum corneum lipids and stratum corneum lipid substitution therapy. Skin Pharmacol Physiol. 2015;28(1):42–55.

    CAS  PubMed  Google Scholar 

  6. Mlitz V, Latreille J, Gardinier S, Jdid R, Drouault Y, Hufnagl P, et al. Impact of filaggrin mutations on Raman spectra and biophysical properties of the stratum corneum in mild to moderate atopic dermatitis. J Eur Acad Dermatol Venereol. 2012;26(8):983–90.

    CAS  PubMed  Google Scholar 

  7. van Smeden J, Bouwstra JA. Stratum corneum lipids: their role for the skin barrier function in healthy subjects and atopic dermatitis patients. Curr Probl Dermatol. 2016;49:8–26.

    PubMed  Google Scholar 

  8. van Smeden J, Hoppel L, van der Heijden R, Hankemeier T, Vreeken RJ, Bouwstra JA. LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery. J Lipid Res. 2011;52(6):1211–21.

    PubMed  PubMed Central  Google Scholar 

  9. Mizutani Y, Mitsutake S, Tsuji K, Kihara A, Igarashi Y. Ceramide biosynthesis in keratinocyte and its role in skin function. Biochimie. 2009;91(6):784–90.

    CAS  PubMed  Google Scholar 

  10. Arct J, Majewski S, Leleń-Kamińska K, Stepulak M, Malejczyk M. Biological activity of ceramides and other sphingolipids. Postep Derm Alergol. 2012;29(3/2012):169–75.

  11. Holleran WM, Takagi Y, Uchida Y. Epidermal sphingolipids: metabolism, function, and roles in skin disorders. FEBS Lett. 2006;580(23):5456–66.

    CAS  PubMed  Google Scholar 

  12. Brooks G, Idson B. Skin lipids. Int J Cosmet Sci. 1991;13(2):103–13.

    CAS  PubMed  Google Scholar 

  13. Farwanah H, Wohlrab J, Neubert RH, Raith K. Profiling of human stratum corneum ceramides by means of normal phase LC/APCI-MS. Anal Bioanal Chem. 2005;383(4):632–7.

    CAS  PubMed  Google Scholar 

  14. van Smeden J, Janssens M, Gooris GS, Bouwstra JA. The important role of stratum corneum lipids for the cutaneous barrier function. Biochim Biophys Acta. 2014;1841(3):295–313.

    PubMed  Google Scholar 

  15. Graeve M, Janssen D. Improved separation and quantification of neutral and polar lipid classes by HPLC-ELSD using a monolithic silica phase: application to exceptional marine lipids. J Chromatogr B Anal Technol Biomed Life Sci. 2009;877(20–21):1815–9.

    CAS  Google Scholar 

  16. Gerits LR, Pareyt B, Delcour JA. Single run HPLC separation coupled to evaporative light scattering detection unravels wheat flour endogenous lipid redistribution during bread dough making. LWT Food Sci Technol. 2013;53(2):426–33.

    CAS  Google Scholar 

  17. Homan R, Anderson MK. Rapid separation and quantitation of combined neutral and polar lipid classes by high-performance liquid chromatography and evaporative light-scattering mass detection. J Chromatogr B Biomed Sci Appl. 1998;708(1):21–6.

    CAS  PubMed  Google Scholar 

  18. Cajka T, Fiehn O. Comprehensive analysis of lipids in biological systems by liquid chromatography-mass spectrometry. TrAC Trends Anal Chem. 2014;61:192–206.

    CAS  Google Scholar 

  19. Holleran WM, Takagi Y, Menon GK, Legler G, Feingold KR, Elias PM. Processing of epidermal glucosylceramides is required for optimal mammalian cutaneous permeability barrier function. J Clin Invest. 1993;91(4):1656–64.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Uchida Y, Hara M, Nishio H, Sidransky E, Inoue S, Otsuka F, et al. Epidermal sphingomyelins are precursors for selected stratum corneum ceramides. J Lipid Res. 2000;41(12):2071–82.

    CAS  PubMed  Google Scholar 

  21. Rabionet M, Gorgas K, Sandhoff R. Ceramide synthesis in the epidermis. Biochim Biophys Acta. 2014;1841(3):422–34.

    CAS  PubMed  Google Scholar 

  22. Bouwstra JA, Ponec M. The skin barrier in healthy and diseased state. Biochim Biophys Acta. 2006;1758(12):2080–95.

    CAS  PubMed  Google Scholar 

  23. t’Kindt R, Jorge L, Dumont E, Couturon P, David F, Sandra P, et al. Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry. Anal Chem. 2012;84(1):403–11.

    PubMed  Google Scholar 

  24. Schmitt T, Neubert RHH. State of the art in stratum corneum research: the biophysical properties of ceramides. Chem Phys Lipids. 2018;216:91–103.

    CAS  PubMed  Google Scholar 

  25. Guillard E, Tfayli A, Manfait M, Baillet-Guffroy A. Thermal dependence of Raman descriptors of ceramides. Part II: effect of chains lengths and head group structures. Anal Bioanal Chem. 2011;399(3):1201–13.

    CAS  PubMed  Google Scholar 

  26. Jennemann R, Rabionet M, Gorgas K, Epstein S, Dalpke A, Rothermel U, et al. Loss of ceramide synthase 3 causes lethal skin barrier disruption. Hum Mol Genet. 2012;21(3):586–608.

    CAS  PubMed  Google Scholar 

  27. Coderch L, Lopez O, de la Maza A, Parra JL. Ceramides and skin function. Am J Clin Dermatol. 2003;4(2):107–29.

    PubMed  Google Scholar 

  28. Marekov LN, Steinert PM. Ceramides are bound to structural proteins of the human foreskin epidermal cornified cell envelope. J Biol Chem. 1998;273(28):17763–70.

    CAS  PubMed  Google Scholar 

  29. Rabionet M, Bayerle A, Marsching C, Jennemann R, Grone HJ, Yildiz Y, et al. 1-O-acylceramides are natural components of human and mouse epidermis. J Lipid Res. 2013;54(12):3312–21.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Imbert L, Gaudin M, Libong D, Touboul D, Abreu S, Loiseau PM, et al. Comparison of electrospray ionization, atmospheric pressure chemical ionization and atmospheric pressure photoionization for a lipidomic analysis of Leishmania donovani. J Chromatogr A. 2012;1242:75–83.

    CAS  PubMed  Google Scholar 

  31. Hvattum E, Uran S, Sandbaek AG, Karlsson AA, Skotland T. Quantification of phosphatidylserine, phosphatidic acid and free fatty acids in an ultrasound contrast agent by normal-phase high-performance liquid chromatography with evaporative light scattering detection. J Pharm Biomed Anal. 2006;42(4):506–12.

    CAS  PubMed  Google Scholar 

  32. Abreu S, Solgadi A, Chaminade P. Optimization of normal phase chromatographic conditions for lipid analysis and comparison of associated detection techniques. J Chromatogr A. 2017;1514:54–71.

    CAS  PubMed  Google Scholar 

  33. Ramos RG, Libong D, Rakotomanga M, Gaudin K, Loiseau PM, Chaminade P. Comparison between charged aerosol detection and light scattering detection for the analysis of Leishmania membrane phospholipids. J Chromatogr A. 2008;1209(1–2):88–94.

    CAS  PubMed  Google Scholar 

  34. Zheng L, T’Kind R, Decuypere S, von Freyend SJ, Coombs GH, Watson DG. Profiling of lipids in Leishmania donovani using hydrophilic interaction chromatography in combination with Fourier transform mass spectrometry. Rapid Commun Mass Spectrom. 2010;24(14):2074–82.

    CAS  PubMed  Google Scholar 

  35. Christie WW. Rapid separation and quantification of lipid classes by high performance liquid chromatography and mass (light-scattering) detection. J Lipid Res. 1985;26(4):507–12.

    CAS  PubMed  Google Scholar 

  36. Postle AD, Wilton DC, Hunt AN, Attard GS. Probing phospholipid dynamics by electrospray ionisation mass spectrometry. Prog Lipid Res. 2007;46(3–4):200–24.

    CAS  PubMed  Google Scholar 

  37. Folch J, Ascoli I, Lees M, Meath JA, Le BN. Preparation of lipide extracts from brain tissue. J Biol Chem. 1951;191(2):833–41.

    CAS  PubMed  Google Scholar 

  38. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957;226(1):497–509.

    CAS  PubMed  Google Scholar 

  39. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37(8):911–7.

    CAS  PubMed  Google Scholar 

  40. Motta S, Monti M, Sesana S, Caputo R, Carelli S, Ghidoni R. Ceramide composition of the psoriatic scale. Biochim Biophys Acta. 1993;1182(2):147–51.

    CAS  PubMed  Google Scholar 

  41. Robson KJ, Stewart ME, Michelsen S, Lazo ND, Downing DT. 6-Hydroxy-4-sphingenine in human epidermal ceramides. J Lipid Res. 1994;35(11):2060–8.

    CAS  PubMed  Google Scholar 

  42. Masukawa Y, Narita H, Shimizu E, Kondo N, Sugai Y, Oba T, et al. Characterization of overall ceramide species in human stratum corneum. J Lipid Res. 2008;49(7):1466–76.

    CAS  PubMed  Google Scholar 

  43. Boiten W, Helder R, van Smeden J, Bouwstra J. Selectivity in cornified envelop binding of ceramides in human skin and the role of LXR inactivation on ceramide binding. Biochim Biophys Acta Mol Cell Biol Lipids. 2019;1864(9):1206–13.

    CAS  PubMed  Google Scholar 

  44. van Smeden J, Boiten WA, Hankemeier T, Rissmann R, Bouwstra JA, Vreeken RJ. Combined LC/MS-platform for analysis of all major stratum corneum lipids, and the profiling of skin substitutes. Biochim Biophys Acta. 2014;1841(1):70–9.

    PubMed  Google Scholar 

  45. Cha HJ, He C, Zhao H, Dong Y, An IS, An S. Intercellular and intracellular functions of ceramides and their metabolites in skin (review). Int J Mol Med. 2016;38(1):16–22.

    CAS  PubMed  Google Scholar 

  46. Vávrová K, Kováčik A, Opálka L. Ceramides in the skin barrier. Eur Pharm J. 2017;64 (2):28–35.

    Google Scholar 

  47. Candi E, Schmidt R, Melino G. The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol. 2005;6(4):328–40.

    CAS  PubMed  Google Scholar 

  48. Hamanaka S, Suzuki A, Hara M, Nishio H, Otsuka F, Uchida Y. Human epidermal glucosylceramides are major precursors of stratum corneum ceramides. J Investig Dermatol. 2002;119(2):416–23.

    CAS  PubMed  Google Scholar 

  49. Schmuth M, Man M-Q, Weber F, Gao W, Feingold KR, Fritsch P, et al. Permeability barrier disorder in Niemann–Pick disease: sphingomyelin–ceramide processing required for normal barrier homeostasis. J Investig Dermatol. 2000;115(3):459–66.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank the Ile de France region for funding the purchase of mass spectrometer LTQ-Orbitrap Velos Pro and Mr. Bastien Prost for his technical assistance.

Funding

This work received financial support from the SILAB - Jean PAUFIQUE Corporate Foundation.

Author information

Author notes

  1. Ali Assi and Joudi Bakar contributed equally to this work.

Authors and Affiliations

  1. Lip(Sys)2, Chimie Analytique Pharmaceutique (FKA EA4041 Groupe de Chimie Analytique de Paris-Sud), Université Paris-Sud, Université Paris-Saclay, 92290, Châtenay-Malabry, France

    Ali Assi, Joudi Bakar, Danielle Libong, Elie Sarkees, Arlette Baillet-Guffroy, Rime Michael-Jubeli & Ali Tfayli

  2. SAMM, UMS IPSIT, Université Paris Sud, Université Paris-Saclay, 92290, Chatenay-Malabry, France

    Audrey Solgadi

Authors
  1. Ali Assi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joudi Bakar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Danielle Libong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elie Sarkees
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Audrey Solgadi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Arlette Baillet-Guffroy
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Rime Michael-Jubeli
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ali Tfayli
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rime Michael-Jubeli.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Assi, A., Bakar, J., Libong, D. et al. Comprehensive characterization and simultaneous analysis of overall lipids in reconstructed human epidermis using NPLC/HR-MSn: 1-O-E (EO) Cer, a new ceramide subclass. Anal Bioanal Chem 412, 777–793 (2020). https://doi.org/10.1007/s00216-019-02301-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-019-02301-3

Keywords

Search

Navigation