Characterization of Vernix Caseosa: Water Content, Morphology, and Elemental Analysis

Characterization of Vernix Caseosa: Water Content, Morphology, and Elemental Analysis William L. Pickens,* Ronald R. Warner,² Ying L. Boissy,² Raymond E. Boissy*³ and Steven B. Hoath*³ *The Skin Sciences Institute and Division of Neonatology, Children's Hospital Medical Center, Cincinnati, Ohio, U.S.A.; ²The Procter & Gamble Company, Cincinnati, Ohio, U.S.A.; ³Department of Dermatology, University of Cincinnati Medical Center, Cincinnati, Ohio, U.S.A. Recent studies have prompted interest in the use of revealed ¯attened structures approximately 1±2 mmin epidermal barrier creams as protective bio®lms for thickness with distinct cellular envelopes indicative very low birthweight preterm infants. The key to of differentiated corneocytes. Compared with mature understanding the role of epidermal barrier ®lms is corneocytes in adult stratum corneum, vernix cor- an elucidation of their interaction with water and a neocytes appeared swollen, the density of the keratin basic knowledge of their composition. In this study, ®laments was less, and there was a relative lack of we investigated the morphologic properties and ele- tono®lament orientation. Cryofractured specimens mental composition of the naturally occurring bio- were examined by cryoscanning electron microscopy ®lm, vernix caseosa. This bio®lm is typically lacking with subsequent elemental localization by X-ray in preterm infants and its production coincides in beam analysis. The ®ndings indicate the high water utero with terminal differentiation of the epidermis content of vernix is largely compartmentalized and formation of the stratum corneum. Signi®cantly, within fetal corneocytes. These results are consistent vernix (80.5 6 1.0% H2O) had a much higher water with the novel view of vernix as a ``¯uid phase'' stra- content than other barrier creams (Eucerin: 17.1 6 tum corneum consisting of a hydrophobic lipid 0.6%, Aquaphor: 0.33 6 0.03%, Ilex: 0.19 6 0.02%, matrix with embedded fetal corneocytes possessing petrolatum: 0.03 6 0.01%; all p < 0.05). Phase con- unique biomechanical and water-binding properties. trast microscopy of vernix showed multiple cellular Key words: corneocyte/epidermal barrier/fetal skin. J Invest elements with nucleic ``ghosts'' embedded in a puta- Dermatol 115:875±881, 2000 tive lipid matrix. Transmission electron microscopy rior to birth, the fetal mammal exhibits an orderly preterm human infants (Nopper et al, 1996). These ®ndings support program of epithelial maturation. In order to cope with the hypothesis that preterm infant skin is missing a protective the exigencies of postnatal life, environmental interfaces mantle of barrier lipids. In the term infant, cutaneous lipids with Psuch as the lung, gut, and skin must be functionally and barrier properties include intercorneocyte lamellar lipids synthe- structurally mature. Paramount among the changes that sized by keratinocytes and the naturally occurring bio®lm, vernix occur during the last trimester of human pregnancy is the caseosa. In this report, we focus on characterization of the latter. development of a highly organized stratum corneum that is Vernix caseosa is a lipid-rich material consisting of wax and sterol required for postnatal epidermal barrier function. Many of the esters, squalene, cholesterol, triglycerides, and free sterols structural changes occurring in skin during human fetal develop- (Karkkainen et al, 1965) as well as cellular elements (Agoratos ment have been well documented (Holbrook and Odland, 1975, et al, 1988). To our knowledge, vernix is uniquely human, 1980). The presence of functional barrier competence by the time although other structures, such as the periderm in rodents may play of birth is evident by the ®nding that transepidermal water loss in a similar part in utero (Wickett et al, 1993; Okah et al, 1995b). Other the newborn term infant is lower than steady-state adult values animals, such as sheep, produce lanolin, which also contains wax (Cunico et al, 1977). and sterol esters (Harris et al, 2000); however, unlike vernix, this Preterm delivery results in a markedly immature epidermal sebaceous secretion has not been reported to contain desquamated barrier (Okah et al, 1995a; Rutter, 1996). Attempts have been made corneocytes. In humans, vernix progressively coats the infant in a to reduce transepidermal water loss in preterm infants by the use of cephalocaudal manner during the last trimester of gestation. The hydrophobic arti®cial polymer ®lms, such as polyethylene wraps high squalene and wax ester content in vernix strongly suggests that (Baumgart, 1984) and topical agents (Rutter and Hull, 1981). a signi®cant portion of the lipid content is of sebaceous origin Recently, it was reported that application of a topical barrier cream (Downing and Strauss, 1974). The onset of vernix production containing petrolatum resulted in improved epidermal barrier approximates the time of stratum corneum formation that begins function and a decrease in nosocomial infection in low birthweight anatomically in proximity to the pilosebaceous apparatus (Hashimoto, 1970; Hardman et al, 1999). Manuscript received February 9, 2000; revised June 27, 2000; accepted A review of the literature reveals a number of biochemical for publication August 2, 2000. studies characterizing the lipid content of vernix caseosa (Downing Reprint requests to: Dr. Steven B. Hoath, Skin Sciences Institute, 231 and Greene, 1968; Nicolaides et al, 1972; Wysocki et al, 1981; Bethesda Avenue, Cincinnati, OH 45267-0541. Email: [email protected] Stewart et al, 1982), but a paucity of morphologic characterization 0022-202X/00/$15.00 ´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 875 876 PICKENS ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY and little information on vernix±water interactions. The present microscope. Alternatively, other vernix specimens were placed in 0.25% study reports direct measurement of the water content of vernix RuO4 in a 0.1 M cacodylate buffer for 1 h at 4°C, rinsed brie¯y in 0.1 M caseosa with a comparison with common epidermal barrier creams. cacodylate buffer and then dehydrated through a graded acetone series prior In order to identify the compartmentalization of water within to Epon embedding and overnight polymerization at 65°C. Thin sections were obtained using an ultramicrotome, counterstained with uranyl acetate freshly harvested human vernix, we also provide ultrastructural and lead citrate, and analyzed by transmission electron microscopy in a characterization of vernix by transmission and cryoscanning Philips CM12 at 100 KeV. electron microscopy accompanied by elemental X-ray analysis of carbon and oxygen. Cryoscanning electron microscopy and elemental analysis Freshly harvested vernix was transported from the delivery room to the laboratory MATERIALS ANDMETHODS under amniotic ¯uid. A small specimen was placed on a gold planchet, ¯ash-frozen in liquid nitrogen-cooled liquid ethane, then transferred to Vernix caseosa and standard barrier creams Within minutes of liquid nitrogen. On a cold stage under vacuum, the frozen specimen was delivery, vernix caseosa was gently scraped from the skin surface of fractured to expose a cross-section, coated with a 2 nm layer of gold/ newborn infants with a plastic spoon and stored in air-tight sterile culture palladium at ±110°C, and analyzed in an Hitachi S4500 scanning electron tubes at 4°C until analysis. Vernix collection was approved by the microscope ®tted with an Oxford cryostage thermocontrolled to ±110°C. Institutional Review Board of the University of Cincinnati Medical Image collection was performed with the electron beam acceleration Center. Aquaphor Healing Ointment and Eucerin Cream were voltage set at 2 KeV. X-ray spectra and elemental maps were obtained at a manufactured by Beiersdorf (Norwalk, CT). Ilex Skin Protectant Paste beam acceleration voltage of 4±6 KeV. The X-ray analysis was performed was manufactured by E.R. Squibb (Princeton, NJ). Petrolatum was with a Link Isis energy dispersive spectrophotometer with a thin window manufactured by CVS (Woonsocket, RI). silicon detector. Determination of the volatile content of vernix and standard RESULTS barrier creams Aliquots of freshly harvested vernix (n = 28) and standard barrier creams (n = 10) were thinly coated on to aluminum Determination of water content Specimens of freshly weigh pans and weighed using a Cahn model C-31 electrobalance. The harvested vernix caseosa and other topical barrier creams specimens were transferred to a vacuum chamber and maintained at 23°C routinely used in the newborn nursery were weighed then until constant weights were obtained. Dry weight to wet weight ratios and desiccated under vacuum until constant weights were obtained. the percentage of volatile component(s) were calculated. Dry weight to wet weight ratios and the percent of evaporative Quanti®cation of water content by Karl±Fischer titration Vernix weight loss were determined. Dry weight to wet weight ratios were specimens were obtained from three separate infants immediately after birth 0.19 6 0.01 and 0.83 6 0.02 (mean 6 SD) for vernix and Eucerin, and stored in air-tight containers at 4°C until analysis. Complete extraction of water was achieved by adding 200 mg of each vernix specimen to 25 ml of a solution containing 60% methanol and 40% formamide and incubating the mixture for 24 h at room temperature. A 3 ml aliquot of each extraction solution was removed for determination of water content by Karl±Fischer titration. The titration was performed

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