Variability in the Fatty Acid Composition of Wax Esters from Vernix Caseosa and Its Possible Relation to Sebaceous Gland Activity
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0022_202X/ 82/ 7804 -0291 $02.00/0 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY, 78:291-295, 1982 Vol. 78, No.4 Copyright © 1982 by The Williams & Wilkins Co. Printed ill U.S.A. Variability in the Fatty Acid Composition of Wax Esters from Vernix Caseosa and Its Possible Relation to Sebaceous Gland Activity MARY ELLEN STEWART, PH.D., MARA A. QUINN, B.S., AND DONALD T. DOWNING, PH.D. Marshall Dermatology Research Laboratories, Department of Dermatology, University of Iowa College of Medicine, Iowa City, Iowa, U.S.A. Wax ester/sterol ester (WE/SE) ratios were measured [8]. These authors commented particularly on their observation in saIUples of vernix caseosa lipid obtained from 21 full that, while the unsaturated fatty acids of adult wax esters are term. infants. The mean WEISE ratio was twice as high almost entirely the products of a uniquely cutaneous ~6-d esat in m.ales as in females, suggesting higher average fetal uration process, the unsatw'ated fatty acids of the vernix ca sebUlD production rates in males, but individual values seosa wax esters contained about 12% of species resulting from varied widely and there was considerable overlap be a ~9-desaturation step. Nicolaides et al postulated that the ~9- tween the sexes. type unsatmates are synthesized in fetal sebaceous glands Wax esters (a class of lipid produced only by the se rather than obtained from the circulation. This conclusion baceous glands) were isolated from 6 of the 21 samples apparently was based on the observation that some of these of vernix caseosa lipid and analyzed for fatty acid com ~9-fatty acids were not derivable, by chain extension, from oleic position. The percentages of iso and anteiso branched acid (18:1~9), which is the major circulating monounsaturated saturated fatty acids and of .19-type monounsaturated fatty acid, but instead consisted of palmit oleic acid (16:1~9) and fatty acids were found to be correlated negatively with its extension products. the WEISE ratios of the original lipid samples. Values Nazzaro-Porro et al [5] studied the fatty acid composition of for the percentages of the major .19-type monounsatu skin surface wax esters from individuals of various ages and rates, plotted as a function of WEISE ratios, fell near a reported that fatty acids having the ~9-pattern of unsaturation curve of the shape which would be expected if a limited were higher in the wax esters of prepuberal children and the quantity of .19-type mono unsaturates were available for elderly than in the wax esters of individuals aged 20-65. This sebuIU synthesis, and were diluted with varying amounts observation indicated that there may be a relationship between of A6-type monounsaturates, the amount of the .16-type sebum production rates and the amount of ~9-type fatty acids monounsaturates being proportional to the amount of in wax esters. wax esters synthesized by the sebaceous glands. The The study of NazzaTo-Porro et al [5] included thin-layer results suggested that the rate of sebum production may chromatographic analyses of vernix caseosa lipid. On the basis be the sole determinant of the percentage of .19-type of these analyses, they concluded that there was a clear-cut monounsaturates in sebaceous wax esters and a partial difference between the 2 sexes in the amount of sebum in vernix determinant of the percentage of iso and anteiso caseosa, with males producing much more sebum than females branched saturates. during fetal life. This observation suggested the present study which sought to further examine the relationship between wax ester fatty acid composition and fetal sebaceous gland activity. The activity of human sebaceous glands val"ies at different Since direct gravimetric measurements of fetal sebum produc stages of life in response to varying levels of circulating anclro tion cannot be made, WEI SE ratios were used as a substitute gens. Maximal activity is reached during puberty and main measurement. Our reasons for believing that WEI SE ratios are tained until menopause in women and until approximately age a valid measure of fetal sebum production aTe outlined in th e 70 in men [1,2]. The increase in sebaceous gland activity at "Discussion. " puberty is accompanied by changes in the relative amounts of We were aided in the analysis of the wax esters by a new various lipid classes on the skin sUl"face, especialJy in the method for separating wax esters from sterol esters using ch.ro amounts of wax esters relative to cholesterol and cholesterol matography on' magnesium hydroxide [9]. This adsorbent is esters [3,4]. These changes probably result mainly or entirely similar in its chromatographic properties to magnesia, which from an increase in the relative contribution of sebaceous lipid was used by Nicolaides et al in their analysis of vernix caseosa compared to epidermal lipid. Fw·thermore, there has been lipids (8], but it has somewhat better resolving power for the speculation that androgenic stimulation may alter the fatty acid sepal"ation of wax esters from sterol esters. Although silica gel composition of the lipids produced by the sebaceous glands has been used also as an adsorbent for isolating wax esters [3,5], [3,5]. Since skin surface trigJycerides and free fatty acids are in oux experience skin surface wax ester and sterol ester bands mixtures of sebaceous and epidermal contributions, efforts to invariably overlap on silica gel. demonstrate changes in fatty acid composition depend mainly on results obtained with wax esters, which are purely sebaceous MATERIALS AND METHODS [6,7]. Thorough analyses of the fatty acid composition of wax esters Vernix Caseosa Lipid from the skin surface of an adult male and from the vernix Vernix caseosa was collected from the skin surface of 21 full -term, caseosa of a male infant have been reported by Nicolaides et al healthy infants using gauze sponges or tongue depressors. Samples which appeared to be excessively contaminated with blood were dis Manuscript received May 26, 1981; accepted for pUblication Septem carded. When the lipid extracts were analyzed chromatographicall y (see next paragraph) some were found to be contaminated with a ber 12, 1981. This work was supported in part by U.S. Publi c H ealth Ser vice nonpolar, non lipid material, whose source we could not identify. K-Y jelly was checked and exonerated. The material had a lower chromato grant number AM22083. graphic mobility than the lipid classes in which we were interested and Reprint requests to: Mary Ellen Stewart, Ph.D., 270 Medical Labo ratories, University ofIowa Coll ege of Medicine, Iowa City, Iowa 52242. did not interfere with our analyses. Abbreviations: WEISE Ratio HPLC: high pressure liquid chromatography TLC: thin layer chromatography 100 f.Lg samples of vernix caseosa lipid were applied to 6 mm lanes WEISE: wax esters/sterol esters scored in 0.25-mm thick layers of silica gel G spread on 20 x 20 cm thin- 291 292 STEWART, QUINN, AND DOWNING Vol. 78, No. 4 layer chromatography (TLC) plates. The plates were qeveloped to the Associates, Milford, MA). A 10-50 mg sample of monoesters was top with hexane and then with toluene. The developed chromatograms injected into the system and eluted from the co lumn with 2% ethyl were charred by spraying with 50% aqueous sulfuric acid and then acetate in hexane flowing at 19.8 rnl/ min. The complete separation heating slowly to 220 0 on a hot plate covered with a 20 x 20 em required about 40 min. aluminum slab. When they had cooled, the charred chromatograms were quantified [10] using a photodensitometer (Clifford 445, Corning Fatty Acid Composition of Wax Esters Medical Products, Medfield, MA) . The overlapping wax ester and sterol The wax esters were saponified by heating to 70 0 for 2 hr with IN ester peaks were triangulated manually, a conection factor was applied KOH in 20:1 methanol-water and the liberated fatty acids were meth to compensate for the nonlinearity of photodensitometer response [11], ylated by adding an excess of BCl:,-methanol directly to the saponifi and the WEISE ratio was calcul ated. cation mixture. Methyl esters were isolated by TLC on 1-mm thick layers of silica gel G developed with toluene. The methyl esters were Isolation of Wax Esters then separated by degree of unsaturation on 0.5 mm layers of silica gel A wax ester + sterol ester (monoester) fraction was separated from H-10% AgNO" developed twice in toluene. The amounts of saturated other vernix caseosa lipid classes by preparative TLC on 1 mm thick monounsaturated, and diunsaturated methyl esters recovered wer~ layers of silica gel G developed as for the analytical plates described estimated by TLC on silica gel G of aliquots of each fraction. Known above. Bands were detected by spraying the plates with a 0.2% solution amounts of methyl oleate were run on the same plate for comparison of 2',7' -dichlorofluorescein in ethanol and subsequent viewing of the of photodensitometric peak areas. The methyl ester fractions from plates under ultraviolet light. The area of silica ge l containing the argentation TLC were analyzed on a 4 mm x 6 ft co lu mn packed with monoesters was scraped from the plate and the lipid was eluted from 10% DEGS installed in an F&M 402 gas chromatograph (Hewlett the adsorbent with ether. Packard Co.) . Peaks were integrated with an electronic integrator Wax esters were separated from sterol esters by chromatography on (Columbia Scientific Industries, Austin, TX, model 204). Structures magnesium hydroxide [9]. A 120 x 2 cm high pressure liquid chroma were identified by plots of log (retention time) versus probable number tography (HPLC) column packed with magnesium hydroxide was used of carbons and by reference to Nicolaides's elu cidation of the structures in conjunction with 2 HPLC pumps connected in parallel, an injector, of the branched fatty acids of vernix caseosa nn and a refractive index detector (all HPLC equipment from Waters To measure the relative amounts of the various double-bond posi tional isomers in the monounsaturates, 1-5 mg samples of monounsat urates were first fractionated by chain length on a 20 ft x 7 mm copper TABLE I.