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

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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. E. V. Truter, Textile Chemical * Mean ± standard deviation. Laboratory, Leeds, England, for these acids. t Number of different samples analyzed indicated in parenthe . S 4 Authors are grateful to Dr. R. P. Hansen, Department of Scientific and Industrial Research, Wellington, New Zealand, ses. @ for a sampleof this acid. PP post-plucking of hair; 4-, 12-, and 22-day PP male and @ ‘Dr. A. W. Weitkamp, American Oil Co., Whiting, Indiana, 22-dayfemale. graciously supplied these acids. §PMC= applications of methylcholanthrene. 6 Hormel Foundation, Austin, Minnesota. # Sq.-ca. —squamous-cell carcinoma.

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TABLE 2 FATTY ACID COMPOSITION (PER CENT OF TOTAL) OF MONO- AND DIGLYCERIDE FRACTION OF MOUSE EPIDERMIS4

aPIDERMISt4-, oi

ACIDNAiuzz 12-,@,and22-day 12-@@,1@n@22-day PMC* * (12:0 (5)4-. (7)PMCI ()18-24 ()sq.-@.

±0.07 ±0.2 ±0.2 ±0.04 ±0.1 14:0 0.5 ±0.17 0.5 ±0.2 0.6 ±0.2 0.5 ±0.1 0.9 ±0.3 16:0 9.1 d 1.4 10.0± 1.7 10.4± 1.0 8.5 ± 0.7 14.3 h 3.4 16:1 2.9 ±0.7 3.0 ±0.2 2.8 ± 1.0 2.7 ± 0.3 4.2 E 1.6 18:0 2.1 ±0.4 1.8 ±0.4 6.0 ±1.1 4.0 ± 0.7 8.9 E 1.7 18:1 25.0 ±2.6 35.5 ± 2.1 24.2 ±4.2 24.3 ±3.5 22.8 ±3.1 18:2 35.5 ± 2.0 37.1 ± 2.2 29.0 ±2.3 26.6 ±2.8 21.2 ± 8.5 20:0 0.9 ±0.3 0.5 ±0.2 1.4 ±0.2 1.3 ±0.3 1.2 ±0.3 20:1 1.3 ±0.6 0.7 ±0.4 1.3 ±0.2 1.2 ±0.5 1.0 ±0.2 Iso 20:0 0.6 ± 0.5 0.8@f 1.3 ±0.8 1.0 ±0.4 7.2 1 3.3 @ 22:0 1 to 1 to 3.7 ±0.7 5.5 ±0.8 3.1 ± 0.8 24:00.2 20.8 ±3.50.2 8.6 ±1.90.4 18.6 ±3.00.2 24.0 ±3.60.3 14.7 ±3.9

4 Mean ± standard deviation of the mean. t Number of different samples analyzed indicated in parentheses. @ PP = post-plucking of hair. §PMC= painted with methylcholanthrene. #Sq.-ca.=squamous-cellcarcinoma. ¶Only detected in one sample. 4* Behenic acid peak was poorly defined in most of these samples; values are approximate.

employed. Also, recoveries from standard mixtures were suggests that peak 8 (Chart 1) may be due in part to the found to be acceptable. For example, in a standard presence of 17 : 1 fatty acid methyl ester, estimated as such mixture of the esters of palmitic, stearic, arachidic, and in Table 1. That the fatty acids corresponding to the behenic acids (each at a concentration of 25 per cent) numbers given in Charts 1 and 2, and also from similar the recovery of each was, respectively, 24.6 ±0.8, 25.3 ± data on the Apiezon M column, are probably correct was 0.1, 25.1 ±0.3, and 24.4 ±1.2 per cent; in a standard ascertained by determining the retention values (16:0 = mixture of the esters of palmitic, stearic, oleic, and linoleic 1.0) on both columns (4). The retention values for the acids (each at a concentration of 25 per cent) the re acid methyl esters from myristic through isoarachidic covery of each was, respectively, 25.2 ±0.4, 24.2 ±0.1, agreed relatively well with those given by Burchfield et 25.6 ± 0.4, and 24.9 ± 0.4 per cent; and in a standard at. (1) at 197°C. but deviated from their values for acids mixture (NIH) of myristic (25 per cent), palmitic (10 per above arachidic as the temperature differences became cent), and stearic (65 per cent), the recovery of each was, accentuated. The Apiezon M column was run at 210°C., respectively, 25.1 ± 1.2, 10.2 ± 0.5, and 64.6 ± 2.1 per since even at this temperature behenic acid had a reten cent. The areas under the curves were measured with a tion time of 160 minutes, and above 210°C. bleeding oc Disc Integrator. Methyl esters of the standard fatty curred. acids were made with a 10 per cent solution of BF3 in methanol by the procedure of Metcalfe €1at.(16). Reduc RESULTS AND DISCUSSION tion of the unsaturated fatty acid methyl esters was The fatty acid composition of the sterol-wax ester frac accomplished with the Adam catalyst and hydrogen by tion of normal epidermis in various stages of growth, of the micro method of Farquhar et at. (7). hyperplasia (early and late), and of the carcinomas is Separation of the methyl esters of the sterol-wax ester given in Table 1. Each of these analyses represents dii fraction from the carcinomas on an ethylene glycol ferent batciw@softissue obtained concurrently over a period adipate column is shown in Chart 1, where the numbers 1— of 6 months to a year. The 4-, 12-, and 22-day post 19 correspond to those of the fatty acids, indicated in the plucking male and 22-day post-plucking female epidermal legend of this chart. That the fatty acid methyl ester sterol-wax ester fractions showed no appreciable differ No. 6 contained palmitoleic acid ; No. 10, oleic acid ; No. ences in fatty acid composition, so they were combined 14, a C20 unsaturated acid; and No. 19, a C22 unsaturated as normal. This fraction of mouse epidermis has a rather acid was confirmed by reduction, respectively, of pal complex fatty acid composition in that some of the iso mitoleic to palmitic, oleic to stearic, 20 : 1 to arachidic, acids, anteiso acids, odd-numbered acids, methyl 11- and 22 : 1 to behenic acids (Chart 2). The changes in eicosanoic, and enucic as well as the common fatty acids peak area are obvious for each of these reductions. The are present. The fatty acid composition of the sterol-wax inverse relationship between the areas of peaks No. 7 and ester fraction of early and late hyperplastic epidermis is No. 8 on Charts 1 and 2, respectively, together with essentially the same as that of normal epidermis, except changes in the per cent of 17 :0 before and after reduction, for some increase in the linoleic acid content in hyper

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1964 American Association for Cancer Research. CARRUTHERS AND HEININc@—Fatty Acid Compo&ion of Mouse Epidermis 1011 plastic epidermis. However, salient differences exist in and between Ehrlich ascites carcinoma cells and several the fatty acid composition of this fraction isolated from normal tissues of mice. Veerkamp et at. (18) also demon the carcinomas as compared with normal and hyperplastic strated that the neutral lipid fractions of different animals epidermis as follows. There is an increase in palmitic (mouse, rat, rabbit, pig, horse, sheep, and ox) possessed and stearic, and a decrease in palmitoleic, arachidic, henei animal specificity in that their fatty acid composition dif cosanoic, isobehenic, behenic, methyl 11-eicosanoic, and fered from animal to animal but resembled one another erucic acids. However, the most significant difference is for the different tissues of one animal. in the increase in unsaturation of the sterol-wax ester REFERENCES fraction of the carcinomas (71 per cent) as compared with 60 per cent for the epidermis. Much of this is in part 1. BURCEFIELD,H.P., ANDSTORRS,E. E. In Biochemical Appli because of unsaturation due to an increase in the linoleic cations of Gas Chromatography. New York, Academic Press, acid level and to the appearance of arachidonic acid which, 1962, p. 549. 2. CARRUTHERS, C. The Fatty Acid Composition of Dermal if present in the epidermal fraction, is there in small and Epidermal Triglycerides and Phosphatides in Mouse amounts. These differences are amplified in Charts 1 Skin during Normal and Abnormal Growth. Cancer Res., and 2. 22:294—98,1962. The fatty acid composition of the mono- and diglyceride 3. . Influence of Sex and Hair Growth Cycle on Lipid Composition of Mouse Epidermis. Proc. Soc. Exp. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1964 American Association for Cancer Research. The Fatty Acid Composition of the Sterol-Wax Ester and Mono- and Diglyceride Fractions of Mouse Epidermis Undergoing Normal and Abnormal Growth Changes

C. Carruthers and A. Heining

Cancer Res 1964;24:1008-1011.

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