Abstract
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.
Highlights
We report on nonhydroxylated 1-O-acylceramides (1-O-ENSs) discovered during a search for unknown lipids in vernix caseosa
Infusion experiments using an ESI source operated in the negative ion mode revealed signals consistent with sphinganine, sphingosine, and other sphingoid bases (Fig. 3A)
High-temperature GC/MS and direct-probe EI-MS showed a number of mostly saturated fatty acid methyl ester (FAME) with 8–32 carbons having straight and methyl-branched chains, but no hydroxyl group
Summary
Materials Vernix caseosa (1–2 g) was collected from healthy newborn subjects delivered at full term (gestation weeks 39–42) immediately after the delivery. Lipids were separated on glass plates (60 × 76 mm) coated with silica gel (60 G for TLC; Merck Darmstadt, Germany) using the mobile phase of hexane:diethyl ether (93:7 by volume). F-1 was reconstituted in chloroform:methanol (2:1 by volume) at a concentration of 30 mg/ml and separated on glass plates (90 × 120 mm) coated with silica gel. The APCI vaporizer and heated capillary temperatures were set to 270°C and 170°C, respectively Nitrogen served as both the sheath and auxiliary gas at a flow rate of 15 and 17 arbitrary units, respectively. The sample for direct-infusion APCI-MS was dissolved in chloroform (250 g/ml) and delivered by a syringe pump (3 l/min) to a low-dead-volume T-piece, where it was mixed with acetonitrile, flowing at 200 l/min into the APCI source.
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