Vernix caseosa, the waxy substance that coats the skin of newborn babies, has an extremely complex lipid composition. We have explored these lipids and identified nonhydroxylated 1-O-acylceramides (1-O-ENSs) as a new class of lipids in vernix caseosa. These ceramides mostly contain saturated C11-C38 ester-linked (1-O) acyls, saturated C12-C39 amide-linked acyls, and C16-C24 sphingoid bases. Because their fatty acyl chains are frequently branched, numerous molecular species were separable and detectable by HPLC/MS: we found more than 2,300 molecular species, 972 of which were structurally characterized. The most abundant 1-O-ENSs contained straight-chain and branched fatty acyls with 20, 22, 24, or 26 carbons in the 1-O position, 24 or 26 carbons in the N position, and sphingosine. The 1-O-ENSs were isolated using multistep TLC and HPLC and they accounted for 1% of the total lipid extract. The molecular species of 1-O-ENSs were separated on a C18 HPLC column using an acetonitrile/propan-2-ol gradient and detected by APCI-MS, and the structures were elucidated by high-resolution and tandem MS. Medium-polarity 1-O-ENSs likely contribute to the cohesiveness and to the waterproofing and moisturizing properties of vernix caseosa.

Department of Analytical Chemistry Faculty of Science Charles University CZ 128 43 Praha 2 Czech Republic

Department of Concrete and Masonry Structures Faculty of Civil Engineering Czech Technical University Prague CZ 166 29 Praha 6 Czech Republic

Department of Obstetrics and Gynecology 1st Faculty of Medicine Charles University and General University Hospital Prague CZ 128 00 Praha 2 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences CZ 166 10 Praha 6 Czech Republic

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Haake A., Scott G. A., and Holbrook K. A.. 2001. Structure and function of the skin: overview of the epidermis and dermis. In The Biology of the Skin. R. K. Freinkel and D. T. Woodley, editors. Parthenon Publishing Group, New York, NY. 19–45.

Menon G. K. 2015. Skin basics; structure and function. In Lipids and Skin Health. A. Pappas, editor. Springer International Publishing AG, Cham, Switzerland. 9–23.

Visscher M. O., and Narendran V.. 2014. Neonatal infant skin: development, structure and function. Newborn Infant Nurs. Rev. 14: 135–141.

Visscher M., and Narendran V.. 2014. The ontogeny of skin. Adv. Wound Care (New Rochelle). 3: 291–303. PubMed PMC

Hoath S. B., Pickens W. L., and Visscher M. O.. 2006. The biology of vernix caseosa. Int. J. Cosmet. Sci. 28: 319–333. PubMed

Kaerkkaeinen J., Nikkari T., Ruponen S., and Haahti E.. 1965. Lipids of vernix caseosa. J. Invest. Dermatol. 44: 333–338. PubMed

Fu H. C., and Nicolaides N.. 1969. The structure of alkane diols of diesters in vernix caseosa lipids. Lipids. 4: 170–175. PubMed

Ansari M. N. A., Fu H. C., and Nicolaides N.. 1970. Fatty acids of the alkane diol diesters of vernix caseosa. Lipids. 5: 279–282. PubMed

Nicolaides N., Ansari M. N. A., Fu H. C., and Rice G. R.. 1972. The fatty acids of wax esters and sterol esters from vernix caseosa and from human skin surface lipid. Lipids. 7: 506–517. PubMed

Stewart M. E., Quinn M. A., and Downing D. T.. 1982. Variability in the fatty acid composition of wax esters from vernix caseosa and its possible relation to sebaceous gland activity. J. Invest. Dermatol. 78: 291–295. PubMed

Tollin M., Bergsson G., Kai-Larsen Y., Lengqvist J., Sjövall J., Griffiths W., Skúladóttir G. V., Haraldsson A., Jörnvall H., Gudmundsson G. H., et al. . 2005. Vernix caseosa as a multi-component defence system based on polypeptides, lipids and their interactions. Cell. Mol. Life Sci. 62: 2390–2399. PubMed PMC

Rissmann R., Groenink H. W. W., Weerheim A. M., Hoath S. B., Ponec M., and Bouwstra J. A.. 2006. New insights into ultrastructure, lipid composition and organization of vernix caseosa. J. Invest. Dermatol. 126: 1823–1833. PubMed

Míková R., Vrkoslav V., Hanus R., Háková E., Hábová Z., Doležal A., Plavka R., Coufal P., and Cvačka J.. 2014. Newborn boys and girls differ in the lipid composition of vernix caseosa. PLoS One. 9: e99173. PubMed PMC

Šubčíková L., Hoskovec M., Vrkoslav V., Čmelíková T., Háková E., Míková R., Coufal P., Doležal A., Plavka R., Cvačka J., et al. . 2015. Analysis of 1,2-diol diesters in vernix caseosa by high-performance liquid chromatography - atmospheric pressure chemical ionization mass spectrometry. J. Chromatogr. A. 1378: 8–18. PubMed

Háková E., Vrkoslav V., Míková R., Schwarzová-Pecková K., Bosáková Z., and Cvačka J.. 2015. Localization of double bonds in triacylglycerols using high-performance liquid chromatography/atmospheric pressure chemical ionization ion-trap mass spectrometry. Anal. Bioanal. Chem. 407: 5175–5188. PubMed

Kalužíková A., Vrkoslav V., Harazim E., Hoskovec M., Plavka R., Buděšínský M., Bosáková Z., and Cvačka J.. 2017. Cholesteryl esters of ω-(O-acyl)-hydroxy fatty acids in vernix caseosa. J. Lipid Res. 58: 1579–1590. PubMed PMC

Oku H., Mimura K., Tokitsu Y., Onaga K., Iwasaki H., and Chinen I.. 2000. Biased distribution of the branched-chain fatty acids in ceramides of vernix caseosa. Lipids. 35: 373–381. PubMed

Hoeger P. H., Schreiner V., Klaassen I. A., Enzmann C. C., Friedrichs K., and Bleck O.. 2002. Epidermal barrier lipids in human vernix caseosa: corresponding ceramide pattern in vernix and fetal skin. Br. J. Dermatol. 146: 194–201. PubMed

Futerman A. H. 2002. Ceramide Signaling (Molecular Biology Intelli­gence Unit 21). Kluwer Academic/Plenum Publishers, New York, NY.

Leray C. 2012. Simple lipids with two different components. In Introduction to Lipidomics: From Bacteria to Man. CRC Press, Boca Raton, FL. 169–209.

Meckfessel M. H., and Brandt S.. 2014. The structure, function, and importance of ceramides in skin and their use as therapeutic agents in skin-care products. J. Am. Acad. Dermatol. 71: 177–184. PubMed

Borodzicz S., Rudnicka L., Mirowska-Guzel D., and Cudnoch-Jedrzejewska A.. 2016. The role of epidermal sphingolipids in dermatologic diseases. Lipids Health Dis. 15: 13. PubMed PMC

Moore D. J., and Rawlings A. V.. 2017. The chemistry, function and (patho)physiology of stratum corneum barrier ceramides. Int. J. Cosmet. Sci. 39: 366–372. PubMed

Stewart M. E., and Downing D. T.. 1995. Free sphingosines of human skin include 6-hydroxysphingosine and unusually long-chain dihydrosphingosines. J. Invest. Dermatol. 105: 613–618. PubMed

Farwanah H., Pierstorff B., Schmelzer C. E., Raith K., Neubert R. H., Kolter T., and Sandhoff K.. 2007. Separation and mass spectrometric characterization of covalently bound skin ceramides using LC/APCI-MS and nano-ESI-MS/MS. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 852: 562–570. PubMed

Pruett S. T., Bushnev A., Hagedorn K., Adiga M., Haynes C. A., Sullards M. C., Liotta D. C., and Jr. Merrill A. H.. 2008. Biodiversity of sphingoid bases (“sphingosines”) and related amino alcohols. J. Lipid Res. 49: 1621–1639. PubMed PMC

Masukawa Y., Narita H., Sato H., Naoe A., Kondo N., Sugai Y., Oba T., Homma R., Ishikawa J., Takagi Y., et al. . 2009. Comprehensive quantification of ceramide species in human stratum corneum. J. Lipid Res. 50: 1708–1719. PubMed PMC

Rabionet M., Gorgas K., and Sandhoff R.. 2014. Ceramide synthesis in the epidermis. Biochim. Biophys. Acta. 1841: 422–434. PubMed

Ponec M., Weerheim A., Lankhorst P., and Wertz P.. 2003. New acylceramide in native and reconstructed epidermis. J. Invest. Dermatol. 120: 581–588. PubMed

Masukawa Y., Narita H., Shimizu E., Kondo N., Sugai Y., Oba T., Homma R., Ishikawa J., Takagi Y., Kitahara T., et al. . 2008. Characterization of overall ceramide species in human stratum corneum. J. Lipid Res. 49: 1466–1476. PubMed

Rabionet M., Bayerle A., Marsching C., Jennemann R., Gröne H. J., Yildiz Y., Wachten D., Shaw W., Shayman J. A., and Sandhoff R.. 2013. 1-O-acylceramides are natural components of human and mouse epidermis. J. Lipid Res. 54: 3312–3321. PubMed PMC

van Smeden J., Boiten W. A., Hankemeier T., Rissmann R., Bouwstra J. A., and Vreeken R. J.. 2014. Combined LC/MS-platform for analysis of all major stratum corneum lipids, and the profiling of skin substitutes. Biochim. Biophys. Acta. 1841: 70–79. PubMed

Wertz P. W., Madison K. C., and Downing D. T.. 1989. Covalently bound lipids of human stratum corneum. J. Invest. Dermatol. 92: 109–111. PubMed

Uchida Y., Hara M., Nishio H., Sidransky E., Inoue S., Otsuka F., Suzuki A., Elias P. M., Holleran W. M., and Hamanaka S.. 2000. Epidermal sphingomyelins are precursors for selected stratum corneum ceramides. J. Lipid Res. 41: 2071–2082. PubMed

van Smeden J., Hoppel L., van der Heijden R., Hankemeier T., Vreeken R. J., and Bouwstra J. A.. 2011. LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery. J. Lipid Res. 52: 1211–1221. PubMed PMC

Jia Z. X., Zhang J. L., Shen C. P., and Ma L.. 2016. Profile and quantification of human stratum corneum ceramides by normal-phase liquid chromatography coupled with dynamic multiple reaction monitoring of mass spectrometry: development of targeted lipidomic method and application to human stratum corneum of different age groups. Anal. Bioanal. Chem. 408: 6623–6636. PubMed

Boiten W., Absalah S., Vreeken R., Bouwstra J., and van Smeden J.. 2016. Quantitative analysis of ceramides using a novel lipidomics approach with three dimensional response modelling. Biochim. Biophys. Acta. 1861: 1652–1661. PubMed

Masukawa Y., Tsujimura H., and Narita H.. 2006. Liquid chromatography-mass spectrometry for comprehensive profiling of ceramide molecules in human hair. J. Lipid Res. 47: 1559–1571. PubMed

Kauhanen D., Sysi-Aho M., Koistinen K. M., Laaksonen R., Sinisalo J., and Ekroos K.. 2016. Development and validation of a high-throughput LC-MS/MS assay for routine measurement of molecular ceramides. Anal. Bioanal. Chem. 408: 3475–3483. PubMed

Vietzke J. P., Brandt O., Abeck D., Rapp C., Strassner M., Schreiner V., and Hintze U.. 2001. Comparative investigation of human stratum corneum ceramides. Lipids. 36: 299–304. PubMed

Fillet M., Van Heugen J. C., Servais A. C., De Graeve J., and Crommen J.. 2002. Separation, identification and quantitation of ceramides in human cancer cells by liquid chromatography-electrospray ionization tandem mass spectrometry. J. Chromatogr. A. 949: 225–233. PubMed

Hsu F. F., and Turk J.. 2002. Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization. J. Am. Soc. Mass Spectrom. 13: 558–570. PubMed

Raith K., Farwanah H., Wartewig S., and Neubert R. H. H.. 2004. Progress in the analysis of Stratum corneum ceramides. Eur. J. Lipid Sci. Technol. 106: 561–571.

t’Kindt R., Jorge L., Dumont E., Couturon P., David F., Sandra P., and Sandra K.. 2012. Profiling and characterizing skin ceramides using reversed-phase liquid chromatography-quadrupole time-of-flight mass spectrometry. Anal. Chem. 84: 403–411. PubMed

Wu Z., Shon J. C., Lee D., Park K. T., Park C. S., Lee T., Lee H. S., and Liu K. H.. 2016. Lipidomic platform for structural identification of skin ceramides with α-hydroxyacyl chains. Anal. Bioanal. Chem. 408: 2069–2082. PubMed

Wu Z., Shon J. C., Kim J. Y., Cho Y., and Liu K. H.. 2016. Structural identification of skin ceramides containing ω-hydroxyacyl chains using mass spectrometry. Arch. Pharm. Res. 39: 1426–1432. PubMed

Laffet G. P., Genette A., Gamboa B., Auroy V., and Voegel J. J.. 2018. Determination of fatty acid and sphingoid base composition of eleven ceramide subclasses in stratum corneum by UHPLC/scheduled-MRM. Metabolomics. 14: 69. PubMed

Farwanah H., Nuhn P., Neubert R., and Raith K.. 2003. Normal-phase liquid chromatographic separation of stratum corneum ceramides with detection by evaporative light scattering and atmospheric pressure chemical ionization mass spectrometry. Anal. Chim. Acta. 492: 233–239.

Farwanah H., Wohlrab J., Neubert R. H. H., and Raith K.. 2005. Profiling of human stratum corneum ceramides by means of normal phase LC/APCI-MS. Anal. Bioanal. Chem. 383: 632–637. PubMed

Stránský K., and Jursík T.. 1996. Simple quantitative transesterification of lipids. 1. Introduction. Eur. J. Lipid Sci. Technol. 98: 65–71.

Motta S., Monti M., Sesana S., Caputo R., Carelli S., and Ghidoni R.. 1993. Ceramide composition of the psoriatic scale. Biochim. Biophys. Acta. 1182: 147–151. PubMed

Lin M. H., Miner J. H., Turk J., and Hsu F. F.. 2017. Linear ion-trap MSn with high-resolution MS reveals, structural diversity of 1-O-acylceramide family in mouse epidermis. J. Lipid Res. 58: 772–782. PubMed PMC

Vrkoslav V., Urbanová K., and Cvačka J.. 2010. Analysis of wax ester molecular species by high performance liquid chromatography/atmospheric pressure chemical ionisation mass spectrometry. J. Chromatogr. A. 1217: 4184–4194. PubMed

Christie W. W. 1987. The separation of molecular species of glycerolipids. In High-Performance Liquid Chromatography and Lipids: A Practical Guide. Pergamon Press, Oxford. 169–210.

Zábranská M., Vrkoslav V., Sobotníková J., and Cvačka J.. 2012. Analysis of plant galactolipids by reversed-phase high-performance liquid chromatography/mass spectrometry with accurate mass measurement. Chem. Phys. Lipids. 165: 601–607. PubMed

Youssef W., Wickett R. R., and Hoath S. B.. 2001. Surface free energy characterization of vernix caseosa. Potential role in waterproofing the newborn infant. Skin Res. Technol. 7: 10–17. PubMed

Robson K. J., Stewart M. E., Michelsen S., Lazo N. D., and Downing D. T.. 1994. 6-Hydroxy-4-sphingenine in human epidermal ceramides. J. Lipid Res. 35: 2060–2068. PubMed

Okabe H., and Kishimoto Y.. 1977. In vivo metabolism of ceramides in rat brain. Fatty acid replacement and esterification of ceramide. J. Biol. Chem. 252: 7068–7073. PubMed

Stránsky K., Jursík T., and Vítek A.. 1997. Standard equivalent chain length values of monoenic and polyenic (methylene interrupted) fatty acids. J. High Resolut. Chromatogr. 20: 143–158.

Oku H., Yagi N., Nagata J., and Chinen I.. 1994. Precursor role of branched-chain amino acids in the biosynthesis of iso and anteiso fatty acids in rat skin. Biochim. Biophys. Acta. 1214: 279–287. PubMed

Combining Charge-Switch Derivatization with Ozone-Induced Dissociation for Fatty Acid Analysis