Changes in Composition and Structure of Triacylglycerol of Adipose Tissue and Skin from Laying Hens as Influenced by Dietary Fats

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1 Changes in Composition and Structure of Triacylglycerol of Adipose Tissue and Skin from Laying Hens as Influenced by Dietary Fats Akihiro HIRATA*, Tetsuya MASUDA*, Teiji KIMURA* and Yoshiyuki OHTAKE* * College of Agriculture and Veterinary Medicine, Nihon University, , Shimouma, Setagaya-ku, Tokyo 154 Effects of dietary soybean oil, coconut oil, lard and beef tallow on the composition and structure of triacylglycerol (TG) of abdominal adipose tissue and skin lipids of laying hens were studied. Fatty acid compositions of adipose tissue and skin TG from each dietary group fairly well reflected the fatty acid pattern of respective fats fed to hens. The lipids from soybean oil, lard and beef tallow feeding groups comprised the TG of C50, C52 and C54 as the major TG components, and that from coconut oil feeding group contained large quantity of medium chain length TG of C36-C48 in addition to the C50-C54. Species composition of TG was determined by argentation thin-layer chromatography. The lipids from soybean oil group contained more amounts of TG of U3 and SU2 than that from the other dietary groups, and the lipids from coconut oil group were abundant in TG of S3 and S2U. Generally, and C18:0 were preferentially esterified at position 1, and C18:2 was predominated in position 2 of TG from adipose tissue and skin of hens. Short chain fatty acids contained in tissue lipids from coconut oil group were largely distributed in position 3. The most abundant TG in lipids from soybean oil, lard and beef tallow groups was sn-uuu, followed by sn-suu and sn-uus, and major components TG of lipid from coconut oil group were sn-sss, sn- SUS and sn-uss both in adipose tissue and in skin lipids of laying hens. Conclusively, it was recognized that the influence of dietary fats on the composition and structure of TG appeared more markedly in the adipose tissue than in skin lipids of laying hens. The fatty acid composition of chicken tissue has been studied by a number of researchers, and there are extensive evidences that dietary fats influence on the component fatty acids of of body fat in animals is generally a reflection of the dietary fatty acid pattern, although the fatty acid composition never completely duplicates that of diet. This is probably due to the influence of dietary fat on fatty acid synthesis in animal tissues5). In laying hen, the triacylglycerol (TG) fraction of adipose tissue lipid more closely reflected the dietary fatty acid composition than did the corresponding phospholipid fraction6). Abdominal adipose tissue and skin of hen are abundant in lipids which are mostly composed of TG7). Therefore, the effects of dietary fats were more distinct in adipose tissue and skin lipids of laying hens than in other tissue lipids of hens8)9). It is supposed that the changes of fatty acid composition in chicken tissue lipid are necessarily caused by the changes of structure and composition of TG which are comprised in the tissue lipids. But, few informations are available concerning the effects of dietary fats on the TG structure and composition of chicken tissue lipids. The purpose of the study reported herein was to investigate the comparative effects of dietary soybean oil,coconut oil, lard and beef tallow on the TG composition and its molecular structure

2 HIRATA et al.: Triacylglycerols Adipose Tissue and Skin of Laying Hens of abdominal adipose tissue and skin lipids of laying hens. Materials and Methods Treatment of the birds Four groups of five laying White Leghorn hens, 8 to 10 months age, were kept in individual cages and fed semi- purified diet supplemented with 10% of soybean oil, coconut oil, lard or beef tallow for a period of 50 days. Feed and water were supplied to the birds adlibitum during the experimental period. Preparation of TG After 50 days on the experimental diets, hens were fasted overnight and sacrificed, and the abdominal adipose tissues and skins were removed from carcasses for lipid analyses. carried out. Total lipids were extracted from each sample according to the method of BLIGH and DYER10), and were separated into neutral and polar lipid fractions by the procedure of MOERCK and BALL11). Then TG was isolated from each neutral lipid fraction by Unicil (Clarkson Chemical Co.) silicic acid column chromatography. Argentation thin-layer chromatography Each TG sample was fractionated by argentation thin-layer chromatography (AgNO3- TLC) depending on the polarity of the TG. Separation of TG was performed on glass plate mm) containing 10%(w/w) AgNO312), using hexane-ethyl ether-benzene-methanol (70:10: 20:1, by volume) as the developing solvent13). The amount of each TG fraction in separated bands on chromatogram was calculated by reference to internal standard added to each for gas-liquid chromatographic analysis, and TG compositions of those bands were calculated on the basis of their fatty acid compositions, as described previously14). Stereospecific analysis The procedure for the stereospecific analysis of TG was carried out essentially following the scheme proposed by BROCKERHOFF15) and modified by CHRISTIE and MOORE16). Briefly, 1,2(2,3)-diacylglycerols (DG) were prepared from TG by reaction with ethyl magnesium bromide and these were converted synthetically into phenyl phospholipid which were hydrolyzed with phospholipase A2 of snake venom (Trimersurus flavoridis. Wako Pure Chemical Industries, Ltd.)14). The resulting lysophosphatide contained the fatty acid of position 1 of the original TG, and the acids of position 2 was obtained by pancreatic lipase hydrolysis of the TG. The fatty acids of position 3 could be determined by difference from the composition of original TG. Gas-liquid chromatography (GLC) Direct gas-liquid chromatography of TG was carried out on Hitachi Model-163 Gas Chromatograph with an on- column injector equipped with dual column and dual hydrogen flame ionization detectors. The operating conditions of GLC and determining procedure of distribution of various TG types were as the same as described previously17). The fatty acid compositions of lipids were determined by the procedure described previously18), using Shimadzu GC-6A MPE Gas Chromatograph and Shimadzu Chromatopac CR-2A. Results and Discussion Effects of dietary fats on the fatty acid composition of adipose tissue and skin lipids of laying hen The fatty acids compositions of TG from abdominal adipose tissue and skin of laying hens fed diets supplemented with soybean oil, coconut oil, lard or beef tallow were presented in Table 1. TG of adipose tissue and skin from hens fed soybean oil-supplemented diet (soybean oil group) alike were largely composed of unsaturated fatty acids, and had the most amounts of linoleic (C18:2) and linolenic acid (C18:3) among those from the other dietary groups. Adipose tissue and skin TG from hens fed coconut oil (coconut oil group) and soybean oil group had a less amount of oleic acid (C18:1) than those from lard and beef tallow feeding groups. In contrast to soybean oil group, TG of both

3 Table 1 Fatty acid composition of triacylglycerol of abdominal adipose tissue and skin lipids from hens fed fat-supplemented diets (mole %) tissues from coconut oil group contained considerably large amounts of lauric (C12:0) and myristic acid (C14:0), accompanied with less of C18:2 than the other dietary groups. Fatty acids compositions of adipose tissue and skin TG from both lard and tallow groups were rather similar. But, the TG of lard group had little more amount of C18:2 and less C18:0 than that of tallow group, probably attributing to the difference of the fatty acid composition of dietary lard from beef tallow. The results obtained from present study suggested that a portion of ingested fatty acids were deposited directly in adipose tissue and skin of laying hens. C18:2 from dietary source is preferentially deposited in tissue, and the short chain saturated fatty acids as well as long chain fatty acids are utilized in synthesis of tissue lipids. Consequently, it was evaluated that the fatty acid compositions of adipose tissue and skin TG of hens were more close reflection of dietary fatty acid pattern, as referred to previous investigation19) on the hen's egg yolk lipid affected by dietary fats. Effects of dietary treatment of laying hen on the adipose tissue and skin TG species based on carbon number The GLC elution patterns of TG of adipose tissues were shown in Fig. 1, and those of skin were in Fig. 2. Every lipids of adipose tissue and skin from soybean oil, lard and tallow groups showed commonly presence of the TG with acyl carbon number 50, 52 and 54 (C50, C52 and C54) as the major TG components, and concomitant minor TG species of C44, C46, and C48. In contrast to those, both adipose tissue and skin lipids from coconut group showed many peaks of medium chain length TG members such as species of C36-C48 those were constituents of the main TG component in coconut oil17). Distribution of TG types by carbon number in adipose tissue and skin lipids were presented in Table 2. Lipids of both tissues from the soybean oil group contained more amounts of C54 type TG than those from the other three dietary groups, and less of C50 than those from lard and tallow groups. This may be due to

4 HIRATA et al.: Triacylglycerols Adipose Tissue and Skin of Laying Hens Fig. 1 GLC separation of triacylglycerols of abdominal adipose tissue lipids from laying hens fed fat-supplemented diets. the higher level of C18 acid in soybean oil, up to 90% of its fatty acid. Lipids of both adipose tissue and skin from coconut oil group comprised considerably large amounts of medium chain TG (C36-C48). It may be attributed to the incorporation of short chain fatty acids of coconut oil into those tissues of hens, and also to the depression of long chain TG contents, contrary to other dietary lipids. Distribution of TG in lipids from lard group were fairly similar to that from tallow group in the respective tissue lipids, regardless of the difference in the stereospecific structure of TG molecules between lard and beef tallow fed to hens. Effects of dietary treatment of laying hen on the tissue and skin TG species based on unsaturation TG of adipose tissue and skin from each groups were separated by means of AgNO3- TLC procedure depending on the unsaturation of TG. The analytical results were given in Fig. 3. TG samples were usually separated into seven or more bands on the chromatogram, though in the case of adipose tissue from coconut oil group less number of bands were detected with same developing solvent system. Some of the molecular species of TG were separated in individual bands, and sometimes, the mixture of TG species were obtained as a single band on chromatogram20). The composition of those bands containing mixture of TG species were calculated on the basis of their fatty acid composition21). The results of analyses on TG components of adipose tissue and skin were shown in Table 3. Adipose tissue and skin lipids from soybean oil group contained more amounts of monosaturated diunsaturated TG (SU2) and triunsa-

5 Fig. 2 GLC separation of Triacylglycerols of skin lipids from laying hens fed fat-supplemented diets. Table 2 Distribution of triacylglycerol types by carbon number in abdominal apipose tissue and skin lipids from hens fed fat-supplemented diets (Wt.%) turated TG (U3) comprising dienoic or trienoic fatty acids such as SMD, SD2, MD2, SMT and D3, and less amounts of trisaturated TG (S3) and disaturated monounsaturated TG (S2U) such as S2M than those from the other dietary groups. Contrary to the soybean oil group, lipids of coconut oil group had the most amounts of S3 and S2U fractions among the

6 (51) Fig. HIRATA et al.: 3 Argentation Triacylglycerols thin-layer Adipose chromato- diets. 3 of Laying 397 Hens given in Table 4. In TG of adipose tissue and skin lipids from the hens of all groups, C16:0 and C18:0were generally predominated in position 1, and C18:2 was preferentially esterified at position 2 of TG, although, C16:0in adipose tissue lipid from lard group was almost evenly distributed in each position of TG molecule. It was supposed that the even positional distribution of C16:0in Component hens and Skin dietary groups tested in respective tissues. Adipose tissue lipid from lard group had somewhat less amounts of S2M and SM2 than those from tallow group, but the skin lipid TG of the two groups showed comparatively similar values. Above-mentioned results may be attributed to the characteristic incorporation of dietary fatty acids into TG of tissues. And it was estimated that the effects of dietary fatty acids appeared more markedly on the adipose tissue than on the skin lipids. Effects of dietary fats on the fatty acids distribution in TG of adipose tissue and skin lipids of laying hen Adipose tissue and skin TG were subjected to stereospecific analyses to investigate the distribution of fatty acids in position 1, 2 and 3 in TG molecules. The results obtained were grams of adipose tissue and skin TG from hens fed fat-supplemented Table Tissue fed triacylglycerol fat-supplemented species diets of abdominal (mole %) adipose tissue and skin lipids from

7 Table 4 Positional distribution of fatty acids in triacylglycerols of abdominal adipose tissue and skin lipids from hens fed fat-supplemented diets (mole %) adipose tissue TG from lard group was attributed to the dietary lard whose position 2 was occupied largely by C16:0. Short chain fatty acids (C10:0. C12:0 and C14:0 of adipose tissue lipids from coconut oil group were largely presented in position 3 of TG, as the results of the incorporation of dietary fatty acids contained in coconut oil that was composed of the short chain fatty acids on the position 3 of its TG.

8 HIRATA et al.: Triacylglycerols Adipose Tissue and Skin of Laying Hens Effects of dietary fats on stereospecific structure of TG from adipose tissue and skin of laying hen The amounts of each TG type were calculated based on the known fatty acids distributon in intact TG assuming a 1-random, 2-random,3- random arrangement. Results obtained on adipose tissue and skin lipids from each dietary group were given in Table 5. The most abundant TG type in lipids from soybean oil, lard and tallow groups were sn- UUU, followed by sn-uus, though the sum amount of SU2 type (sn-suu, sn-usu and sn- UUS) was more present than that of U3 in those lipids. In contrast to this, the major TG types of coconut oil group were sn-sss, sn-sus and sn-uss in both adipose tissue and skin lipids, and the sn-uuu was fairly less in skin of coconut oil group than in those of the other dietary groups. The sn-uuu, also sn-ssu, sn-suu and sn-usu were the minor TG components in adipose tissue lipid from coconut oil group. A comparison between the findings by Ag NO3-TLC and those by calculation based on the 1-random, 2-random, 3-random distribution was given in Table 6. Considerably good agreements were observed between the amounts Table 5 Predicted triacylglycerol compositions of abdominal adipose tissue and skin lipids from hens fed fat-supplemented diets (mole %) Table 6 Comparison of observed and predicted values of triacylglycerols of abdominal adipose tissue and skin lipids (mole %)

9 found by AgNO3-TLC procedure and the values calculated by random distribution hypothesis, though, some discrepancies were observed among the values obtained from different method on the SU2 fraction of adipose tissue TG from soybean oil group and on the U3 fractions of both adipose tissue and skin TG from coconut oil group. These results suggested that a 1-random, 2-random, 3-random distribution of fatty acids occurred in the course of TG synthesis in adipose tissue and skin of hens on the feeding of lard and tallow. While, on the feeding of soybean and coconut oil, it was supposed that a preferential association of some fatty acids occurred in the course of acylation of glycerols in tissues of laying hens. References 1) MACHLIN, L.J., GORDON, R.S., MARR, J. and POPE, W.S.: Poultry Sci., 41, 1340 (1962). 2) SELL, J.L., CHOO, S.H. and KONDRA, P.A.: Poultry Sci., 47, 1296 (1968). 3) GUENTER, W., BRAGG, D.B. and KONDRA, P.A.: Poultry Sci., 50, 845 (1971). 4) SIM, J.S., BRAGG, D.B. and HODGSON, G.C.: Poultry Sci., 52, 51 (1973). 5) BOTTINO, N.R., ANDERSON, R.E. and REISER, R.: J. Am. Oil Chem. Soc., 42, 1124 (1965). 6) ISAACKS, R.E., DAVIES, R.E., FERGUSON, T.M., REISER, R. and COUCH, J.R.: Poultry Sci., 43, 105 (1964). 7) MARION, J.E. and WOODROOF, J.G.: J. Food Sci., 30, 38 (1965). 8) HIRATA, A., NISHINO, M., KIMURA, T. and OHTAKE, Y.: Nippon Shokuhin Kogyo Gakkaishi, 33, 480 (1986). 9) HIRATA. A., NISHINO, M., KIMURA, T. and OHTAKE, Y.: Nippon Shokuhin Kogyo Gakkaishi, 33, 631 (1986). 10) BLIGH, E.G. and DYER, W.J.: Can. J. Biochem., 37, 911 (1959). 11) MOERCK, K.E. and BALL, Jr., H.R.: J. Food Sci., 38, 978 (1973). 12) WOOD, R. and SNYDER, F.: J. Am. Oil Chem. Soc., 43, 53 (1966). 13) CHRISTIE, W.W. and MOORE, J.H.: Biochim. Biophys. Acta., 210, 45 (1970). 14) OHTAKE, Y.: Jap. J. Zootech. Sci., 54, 179 (1983). 15) BROCKERHOFF, H.: J. Lipid Res., 6, 10 (1965). 16) CHRISTIE, W.W. and MOORE, J.H.: Biochim. Biophys. Acta., 176, 445 (1969). 17) HIRATA, A., MASUDA, T., KIMURA, T. and OHTAKE, Y.: Nippon Shokuhin Kogyo Gakkaishi, 34, 320 (1987). 18) OHTAKE, Y.: Jap. J. Zootech. Sci., 53, 797 (1982). 19) HIRATA, A., NISHINO, M., KIMURA, T. and OHTAKE, Y.: Nippon Shokuhin Kogyo Gakkaishi, 32, 892 (1985). 20) CHRISTIE, W.W. and MOORE, J.H.: Lipids, 4, 345 (1969). (Received Oct. 9, 1986)

10 HIRATA et al.: Triacylglycerols Adipose Tissue and Skin of Laying Hens