The Fatty Acid Composition of the Stero1@Wax Ester and Mono-. and Diglyceride Fractions of Mouse Epidermis Undergoing Normal and Abnormal Growth Changes * C. CARRUTHERS AND A. HEINING (Department of Biochemistry Research, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York) SUMMARY The sterol-wax ester and mono- and diglyceride fractions were obtained by silicic acid chromatography from the total lipid fractions of epidermis, removed from male and female mice 4, 12, and 22 days following plucking of hair, from methylcho lanthrene-treated epidermis (painted 3 or 4 times on alternate week days or for 18 to 24 times on alternate week days for 6-8 weeks, respectively) and from carcinogen induced squamous-cell carcinomas. The sterpi-wax ester fraction of normal and by perplastic epidermis did not differ appreciably, except that the latter contained more oleic acid than did that from normal epidermis. On the other hand, the sterol-wax ester fraction of the carcinomas differed significantly from normal or hyperpiastic epidermis in that it contained more palmitic and stearic, less arachidic, methyl 11- eicosanoic, heneicosanoic, isobehenic, behenic, and enucic acids, and much more linoleic acid than did normal or hyperplastic epidermis. The most significant find ing was that the sterol-wax ester fraction of the carcinomas contained 12 per cent arachidonic acid, which, if present, was at very low levels in the epidermal fraction; this fraction was more unsaturated than that from normal or hyperpiastic epidermis. The mono- and diglyceride fraction from the carcinomas contained more stearic acid than did normal and hyperplastic epidermis. The most interesting difference in this fraction was that, following plucking, female epidermis (4-, 12-, or 22-day) had a low level of lignoceric acid. Previous studies on the lipid composition of mouse MATERIALS AND METHODS epidermis undergoing normal growth changes induced by Swiss mice, 2—4months of age, were used throughout. the hair growth cycle and of this tissue undergoing malig Mice were fed the Derwood and Morris high fat (10 per nant transformation produced by the topical application of cent) diet made by A. E. Staley Company, Decatur, liii methyicholanthrene have indicated that these growth nois. This diet has been used for several years in this changes are associated with alterations in some of the lipid laboratory. Procedures for the preparation of epidermis constituents (2, 3, 5). The investigations of Veerkamp as a function of the hair growth cycle, for painting the et at. (17, 18), KOgl et at. (14), and Figard et a!. (8) have mice with the carcinogen, and for the removal of epidermis also demonstrated that significant differences exist between from dermis and extraction of total lipid have been de the lipid composition of tumors and their homologous scribed (2, 3). Epidermis from 100-200 mice and 20-30 normal tissues. squamous-cell carcinomas, depending on the need, were The purpose of this investigation was to determine the pooled for each analysis. The sterol-wax ester fraction fatty acid composition of the sterol-wax ester and mono (that fraction consisting of stem! fatty acid esters and and diglyceride fractions of mouse epidermis undergoing higher alcohol fatty acid esters) and the mono- and di normal and abnormal growth changes. This approach glyceride fraction were obtained by siicic acid chromatog amplifies and extends our previous research on changes in raphy of the neutral lipid or total lipid portions of the the various lipid constituents in epidermis, and in so tissues (13). These fractions were transesterified with doing helps to determine whether these tissue components methanolic sulfuric acid. The methyl esters thus ob are altered significantly in the process of epidermal carcino tained were separated from the unsaponifiables or other genesis. impurities by column chromatography on siicic acid by the method of Luddy et at. (15). The unsaponifiables * Aided in part by Grant No. CA-06107.02 from the U.S.P.H.S. were eluted from the silicic acid with ethyl ether, recovered, Received for publication January 18, 1964. and weighed. 1008 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1964 American Association for Cancer Research. CARRUTHERS AND HEINING—FaUy Acid Composition of Mouse Epidermis 1009 Lu Lu (I) Cl) z z 0 a-0 a. Cl) U) Lu Li I0 0 I- g C) C) Lu Lu I- I— Lu Lii 0 0 ‘7 3 4 ‘9 20 30 0 I0 20 TIME (mm.) TIME (mm.) CHART 1.—Separation of the fatty acid methyl esters derived from the sterol-wax ester fraction of squamous-cell carcinomas CHART 2.—Separation of the fatty acid methyl esters derived at 195°C.on a 5 ft. X i-in. column containing ethylene glycol from the sterol-wax ester fraction of squamous-cell carcinomas adipate as the partitioning liquid. (1) 14:0; (2) anteiso 15:0; following reduction with hydrogen and Adam catalyst. Same (3)15:0;(4)iso16:0;(5)16:0;(6)16:1;(7)17:0;(8)iso18:0; material and temperature as in Chart 1. (1) 14:0; (2) anteiso 15:0;(3)15:0;(4)iso16:0;(5)16:0;(7)17:0;(8)iso18:0;(9) (9)18:0;(10)18:1;(11)19:0and 18:2;(12)iso20:0;(13)20:0; 18:0;(9A)anteiso19:0;(11)19:0;(12)iso20:0;(13)20:0;(15) (14)20:1;(15)anteiso21:0;(16)21:0;(17)iso22:0and 20:4; anteiso21:0;(16)21:0;(17)iso22:0;(18)22:0. (18) 22:0; (19) 22:1. TABLE 1 The fatty acid composition of the methyl esters was FATTY AcID COMPOSITION (PER CENT OF TOTAL) OF STEROL-WAx determined by gas-liquid chromatography on an Aero ESTER FRACTION OF MOUSE EPIDERMIS graph Hy Fi Model 600 Chromatograph with a hydrogen UNDERGOING CARCINOGENESIS flame ionization detector (4). The polar column, 5 ft. X *in.stainlesssteel,waspackedwith12percentethylene zpinziiasfCombined OP glycol adipate' on 80/100 mesh Gas Chrom P. The non AcmNATIYLE o― PMC@ PMC@ @ polar column of the same dimensions was packed with 15 and PP@ per cent Apiezon M on 80/100 mesh Gas Chrom P. The (5)14:00.5 (14)3 (5)24 (6)Sq.-ca.@@ ethylene glycol adipate column was run at 195°C. and 0.1Anteiso ±0.050.4 ±0.150.4 ±0.030.6 ± the Apiezon M one at 210°C. The methyl esters of the 15:00.30.0415:00.4±0.00.4 ±0.020.4 ±0.10.5 ± fatty acids were dissolved in CS2 at 0.5—1per cent solu 0.3Iso ±0.080.6 ±0.10.6 E 0.20.5 ± tions, and 2—10,@l.were injected per analysis. The identi 0.216:04.115:01.6 ± 0.151.2 ± 0.41.8 ± 0.20.8 ± fication of the fatty acid methyl esters was based upon a 1.216:112.9± 0.35.2 ± 1.04.4 ± 0.69.5 ± comparison of the retention values (16:0 = 1.0) with 1.017:00.9± 1.129.1 ± 1.010.0 ± 1.45.1 ± known fatty acid methyl esters which were : heptadeca 0.117:11.1 ±0.11.2 ±0.40.6 ±0.10.3 ± noic,' pentadecanoic,' nonadecanoic,1 heneicosanoic,' 0.1Iso ± 0.121.0 ± 0.41.2 ± 20-methylheneicosanoic,2 isomi'4'5 12-methyltet 18:00.70.218:02.8 ±0.10.6 ±0.040.6 ±0.10.6 ± radecanoic,5 14-methylhexadecanoic,5 isostearic,3 .B 16- 0.518:123.7±0.121.7 ±0.52.1 ±0.26.6 ± methyloctadecanoic,5 isoarachidic,5 18-methyleicosanoic,5 0.618:22.9± 2.626.1 ± 3.725.9 ± 2.925.3 ± 3.2Anteisol9:01.4 ± 0.78.1 ±1.65.3 ±1.218.3 ± @ isoni@5 myristic,6 palmitoleic,6 stearic,6 0.919:02.3 ±0.61.2 ±0.21.1 ±0.72.2 ± methyl 11-eicosanoic,6 erucic,6 and behenic.6 Identifica 0.2Iso2O:04.6 ±0.71.9 ±0.41.4 ±0.20.4 ± tion was aided in part by plots of the log retention time 0.220:08.7 ± 0.254.6 ± 0.64.9 ± 0.61.3 ± versus the number of carbons in a homologous fatty 0.220:110.8 ±0.388.7 ± 1.08.3 ±0.63.1 ± acid series. Some of the esters at various concentra 0.720:411.9±0.77.7 ±0.410.0 ±1.94.3 ± tions were used to calibrate the GLC apparatus. 1.7Anteiso2l:01.1 ± In this way the detector response was found to be 0.521:03.7± 0.151.1 ± 0.51.7 ± 0.71.2 ± proportional to the concentration of the fatty acids 0.21so22:03.2 ±0.64.4 ±0.83.6 ±0.60.8 h 0.222:02.9 :1: 0.62.8 ±0.63.9 ±0.80.8 ± I Applied Science Laboratories, Inc., State College, Pa. 0.822:19.9 ±0.65.0 ±1.23.1 ±1.21.8 ± 2 A gift from Dr. van Gent, Laboratorium Voor Anorganische ±1.49.0 ±0.89.5 ±1.26.2 ±1.4 En Fysiache Chemie, Leiden, The Netherlands. aAuthors are indebted to Dr.