THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Vol. 44, No. 5 Copyright 1965 by The Williams & Wilkins Co. Printed in U.S.A. LIPIDS OF VERNIX CASEOSA* J. KARKKAINEN, M.D., T. NIKKARI, M.D.t, S. RUPONEN, M.D., AND E. HAAHTI, DR.MED.SCLt Lipids of vernix caseosa of the human fetusHexane (pure, Merck). were quantitatively analyzed first by SchmidMethanol purum, redistilled after treatment with magnesium turnings and iodine. (1). Several authors (2, 3, 4) have later pub-Petroleum spirit (60—80°, Analar). lished more specific data about the general lipidToluene (G. R., Merck). pattern and the fatty acids of the vernix. Our earlier investigations (5) showed a similarity be-. List of Reference Compounds tween the fatty acids of vernix caseosa and thoseFatty acid methyl ester quantitative mixture (H- of seburn and other epidermal lipids, support- 104, Applied Science Laboratories, lot No. 458- ing the hypothesis of the vernix being a product 43). Methyl esters obtained from commercial corn oil. of epidermal secretion. To clarify the questionMyristyl, cetyl, and stearyl alcohols (Applied further we analyzed in tins investigation the Science Laboratories, lots Nos. 76-22, 230-70, lipid composition of normal vernix caseosa using and 230-72, respectively). column chromatography, thin-layer chromatog-c-hydroxy myristyl alcohol was derived from - hydroxymyristic acid using the method de- raphy (TLC), and gas-liquid chromatography scribed by Link e al. (8). (GLC) as principal methods. Squalene (C grade, Calbiochem) Sterols (cholesterol and lathosterol) were ob- MATERIALS AND METHODS tained as a gift from Dr. E. C. Horning, Baylor Samples of vernix caseosa, collected immediately University College of Medicine, Houston, Texas. after birth, were dissolved in chloroform-methanol Column chromatography. Silicic acid (Mallinc- 2:1 (v:v). The lipids were purified according tokrodt, 100 mesh) was sieved to yield 80—100 mesh Folch et al. (6), and stored at 4° under nitrogenmaterial, washed with 3N hydrochloric acid and atmophere as 1% solution in hexane. water, and dried at 150° C. in vacuum for 24 hrs. Free fatty acids were extracted with 0.05%Water was added to give 10% moisture of the aqueous potassium hydroxide in conventional man- support. The amount of silicic acid was chosen to ner. be approximately 30 times the weight of the Alkaline hydrolyses were carried out with 10%sample. Rising concentrations of benzene or ethyl potassium hydroxide in methanol-toluene-waterether in hexane as well as chloroform and methanol 9:1:1 (v: v: v). Screw-cap (teflon-lined) test tubeswere used for elution. The fractions were collected were sealed under nitrogen atmosphere and left atin 10 ml portions. For continuous monitoring of 60° over night. The saponifiable and nonsaponifi-effluents of silicic acid chromatography, an appa- able portions were extracted with ethyl ether-pe-tus devised by Haahti e al. (9, 10) was used. troleum ether 1:4 (v:v). Tubes containing the same fraction were combined, Free and liberated fatty acids were methylatedthe solvents evaporated, and the residue weighed. with diazomethane prepared from "Diazald" (Al-Before further analysis the homogeneity of the drich Chemical Co.). fractions was controlled with TLC. Mono- and dihydric alcohols and hydroxy acids Thin-layer chromatography (TLC). An equip- were acetylated with acetic anhydride accordingment manufactured by Desaga (Heidelberg) was to Link et al. (7). used. The plates were coated with a 0.25 mm layer of Kieselgel G (Merck) or of Aluminum Oxide List of Solvents G (Merck) and dried at 120° for 2 hrs. Sulfuric acid (30%) was mostly used to spray the plates Benzene cryst. (G. R., Merck). for documentation. For preparative purposes the Chloroform (PhF VII 930/62), treated with cal-TLC fractions were made visible with Rhodamine cium chloride, redistilled, and mixed with 1% of6G (BDH) in ethanol. The areas of the support methanol. containing the fractions were scraped off the Ethyl ether (G. R., Merck). plates into centrifuge tubes and extracted three times with chloroform. The extracts were com- This work was supported by the PHS Researchbined, the solvent evaporated and the amount of grant HE-06818 from the National Heart Insitue,recovered material was estimated gravimetrically. Bethesda, Md., U.S.A. Received for publication June 24, 1964. Waxes and sterol esters were separated prepara- * From the Department of Medical Chemistry,tively on aluminum oxide with 5% benzene in pe- University of Turku, Finland. troleum ether as the mobile phase. Fractionation of f Established Investigator of the Finnish Medi-fatty acids and waxes on unsaturation basis was cal Research Council. carried out on thin layer plates of silver nitrate im— 333 334 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY pregnated silica gel (11). The plates were dried inlar microhydrogenation apparatus usually causes darkness first 10 mm. at room temperature anda source of contamination and loss of sample. then 30 mm. at 100°. The identification of frac- Qualitative identification of fatty acids and al- tions on thin layer chromatography plates wascohols was based on relative carbon numbers calcu- based on simultaneous separations of known refer- lated in the conventional manner from a loga- ence compounds. In unclear cases (e.g. diol esters) rithmic plot of retention times of the correspond- the unknown fraction was isolated and identifieding reference compounds against the number of on the basis of chemical and chromatographictheir carbon atoms. The principal scheme for methods. These include transesterification followedqualitative identification or characterization of by TLC analysis of the product (e.g. diols andrecorded esters was as follows: methyl esters), TLC and GLC of the plain and a. The total GLC analysis of the given ester acetylated nonsaponifiables (e.g. diols) giving evi-fraction. dence on the number of hydroxy groups. The b. GLC analysis of a hydrogenated sample of identity was finally confirmed by the synthesis ofthe esters. corresponding compounds from pure reference ma- c. GLC analysis of isolated (AgNO3-TLC) satu- terial as described in detail by Nikkari (12). rated esters. Gas-liquid chromatography (GLC). Gas chro- d—f. GLC of isolated mono-, di- and tri-unsatu- matographic analyses were carried out with arated esters respectively. Barber-Colman M-10 chromatograph with a hy- g. GLO of hydrogenated samples described in drogen flame ionization detector. Nitrogen was 4—7. used as the carrier gas, the columns and conditions Record (a) is used for a general view of the being the following: total sample, and as a reference for the following A 12 ft column of 3% EGSS-X polyester (arecords (b—g). Records (c—f) give the fractions of methyl silicone ethylene glycol succinate co-poly-different unsaturation classes (from zero to three mer, Applied Science Laboratories, State College,double bonds), and the chain length and branch- Pa.) on 100—140 mesh siliconized Gas-Chrom P.ing of these are confirmed by conversion to the Nitrogen pressure was 20 psi at column inlet;corresponding saturated compounds by means of carrier gas flow was 25—35 ml/min. Column tem-hydrogenation (8). peratures were 187° and 153° for fatty acid methyl For quantitative analysis the peaks of the gas esters and 187° for acetylated mono- and dihydricchromatographic records were triangulated, and alcohols and acetylated hydroxy acid methyl esters. the percentages were calculated on the basis of the A 6 ft column of 1% silicone gum SE-30 on 100-surface areas of the triangles. 140 mesh siliconized Gas-Chrom P. Nitrogen pressure was 16 psi; carrier gas flow rate was 40— RESULTS 50 ml/min. Column temperature was 211° for squalene, sterols, dihydric alcohols, hydroxy acid Fig. 1 shows the main lipid fractions of methyl esters, and the total nonsaponifiable mat-vernix caseosa of six subjects as analyzed with ter. TLC. All samples were purified with TLC before gas Gas chromatography of the saponifiable and chromatographic analysis. Catalytic hydrogenation of esters was carriednonsaponifiable portions of unfractionated vernix out as follows: The sample was dissolved in aboutcaseosa lipids as analyzed on EGSS-X at 153° 2 ml methanol in a conical centrifuge tube. Aboutand SE-30 at 211° respectively is presented in 1 mg of platinum oxide catalyst (Matheson Cole-Fig. 2. man & Bell), previously washed with methanol, was added as a methanolic suspension with a Three lipid samples were subjected to silicic pasteur pipette. Hydrogen was then allowed toacid column chromatography (Table I); a bubble through the solution by means of a verytypical elution pattern is seen in Fig. 3. The narrow glass capillary. The hydrogen flow (aboutpercentages of waxes and sterol esters were 2—S ml/min) and the position of the capillary tipdetermined by weighing after their separation were adjusted so that a continuous mixing of the catalyst was obtained. The procedure was carriedon aluminum oxide thin-layer plates as described out in a ventilated hood at room temperature.above. The hydrogenation was allowed to go on for not The column chromatographic fractions of one less than 30 minutes, whereafter the tube wassample (denoted by A in Table I) were analyzed centrifuged at 2000 rpm, the supernatant recovered, in detail with GLC. If not further specified the evaporated under a stream of nitrogen and dis-results later in this paper refer to this sample. solved in hexane. Hydrogenation of known un- Part of the total saponifiable matter was ana- saturated fatty acid methyl esters followed by gas chromatographic analysis showed that thelyzed as described above, on unsaturation basis hydrogenation was complete in 15—20 minutes (12). using thin-layer plates of silica gel impregnated The method seems particularly useful for micro-with silver nitrate. Saturated and monoenoic analytical work with GLC, where the use of regu-acids could be recovered. Di- and polyunsatu- LIPIDS OF VERNIX CASEOSA 335 — e — a SQUALENE • a a • WAXES, STEROL ESTERS S — S DIOL ESTERS II * • TRIGLYCERIDES S S S S FREE STEROLS — — t START, POLAR LIPIDS FIG.