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`822 H. FRICKE, G. GERCKEN, W. SCHREIBER AND J. OEHLENSCHLAGER Fatty acid methyl ester (FAME) of total lipids and individual lipid classes were prepared with 14% boron trifluoride in methanol (18), and fatty acid benzyl esters (FABE) according to Klemrn et al. (19). Trimethylsilylation of sterols was carried out as described by Ballan- tine et al. (20). FAME and FABE were purified by TLC prior to GLC analysis. Separations and identifications were carried out on a polar wall coated (WCOT) open-tubular glass column (25 m) coated with SILAR 10 C (Packard in- struments), temperature programmed from 110 C to 210 C (3 C/min) and on a 50 m fused silica column (WCOT) coated with CP SIL 5, temperature programmed from 100 C to 320 C (3 C/min) using a Packard 428 gas chromato- graph equipped with a FID and a HP 3371 integrator. Helium was used as carrier gas at a flow of 1 ml/min with a split ratio of 100:1. The presence of plasmalogens and alkylglycer- ols was tested subsequent to hydrolysis using the procedure of Pugh et al. (21). GLC/MS analysis of FAME and trimethyl- silyl (TMS) sterols was performed on a HP 5985A quadrupole mass spectrometer, ioniza- tion energy 70 eV, ion source temperature 200 C, column: 25 m WCOT coated with CP SIL 5 (Chrompak), temperature programmed from 140 C to 280 C (4 C/min). Individual FAME, FABE and TMS sterol peaks were identified by co-chromatography with standards, by comparison with calculated equivalent chain length (ECL) values (22) and by mass spectra. To ensure identification of unusual fatty acids, samples were hydrogenated and rechromatographed. For positional analy- sis, cleavage of PC and PE was performed with phospholipase As from Crotalus durissus terrificus (Boehringer, Mannheim). After 24 hr incubation in diethylether and 0.1 M tris-buffer, the reaction mixture was separated by TLC into lysophospholipids and FFA. RESULTS AND DISCUSSION Lipid Content and Lipid Composition The total lipid content and the lipid compo- sition data of the 2 krill samples are given in Table 1. Although different lipid compositions have been published, there is general agreement as to the main lipid classes present in Euphausia superba (3-12). The krill caught in December 1977 has a lower fat content than the krill caught in March 1981. This increase in fat con- tent during the catching season, which co- incides with the sexual maturity (2) of krill, has been shown previously (14). Beginning with a low fat content of approx. 1% on a wet weight basis in November/December, the fat content TABLE 1 Lipid Composition of Antarctic Krill (Euphausia superba Dana) Sample 12/1977 3/1981 Total lipid content (% wet weight) 2.7 + 0.2 6.2 -+ 0.3 Phospholipids Phosphatidylcholine 35.6 -+ 0.1 33.3 -+ 0.5 Phosphatidylethanolamine 6.1 +- 0.4 5.2 + 0.5 Lysophosphatidylcholine 1.5 +- 0.2 2.8 -+ 0.4 Phosphatidylinositol 0.9 + 0.1 1.1 -+ 0.4 Cardiolipin 1.0 -+ 0.4 l Phosphatidic acid 0.6 + 0.4 1.6 (cid:12)9 0.2 Neutral lipids Triacylglycerols 33.3 -+ 0.5 40.4 +- 0.1 Free fatty acids a 16.1 -+ 1.3 8.5 -+ 1.0 Diacylglycerols 1.3 -+ 0.1 3.6 -+ 0.1 Sterols 1.'7 -+ 0.1 1.4 +- 0.1 Monoacylglycerols 0.4 + 0.2 0.9 + 0.1 Others b 0.9 -+ 0.1 0.5 + 0.1 Total 98.9 99.3 Data are expressed as wt % of total lipids and represent means + standard deviation of 3 separate experiments. aprobably mostly artifacts. bTraces of lysophosphatidylethanolamine, phos- phatidylserine, sphingomyelin, glycolipids, sterol es- ters, waxes and carotenoids were detected. increases to approx. 6% in March/April. Euphausia superba is extremely rich in phos- pholipids (I>40% of total lipids) and TG (33 and 40% respectively of total lipids). While the relative content of phospholipids is similar in the 1977 and 1981 samples, the percentages of TG differ somewhat. This is in accordance with the previous results of our laboratories (23), which show that the relative phosphotipid con- centration will not change with varying total lipid contents. In other marine organisms an increase of total lipid content usually is caused by an increase of TG (24). The sterol contents of 1.4% and 1.7% re- spectively of total lipids are in the range which has been reported (2,25) for Krill. These are very low values compared with those of Clarke (3), who found up to 16.9% sterols of total lipids in krill from South Georgia. This differ- ence may be due to the methods. Clarke used densitometry (3) and our laboratory GLC. (cid:12)9 In the 1977 sample the FFA content is about twice that of the 1981 sample. The high value could be caused by the longer storage time of the 1977 sample. A residual lipolytic activity against phospholipids exists even at temperatures of -30 C and below. Samples of LIPIDS, VOL. 19, NO. 11 (1984)
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`ANTARCTIC KRILL LIPIDS 823 the same haul which were cooked on board immediately after hauling and stored under the same conditions showed a FFA content which was much lower, ranging from 1% to 3% of total lipids. This low FFA content of freshly caught krill also was confirmed by Ellingsen (ll). In addition, lysophosphatidylcholine, lyso- phosphatidylethanolamine, phosphatidylinosi- tol phosphatidic acid, cardiolipin and mono- and diacylglycerols were detected, whereas phosphatidylserine, sphingomyelin, glycolipids, wax esters and sterol esters were present only in trace amounts. Wax esters were found by Bottino (8) in the euphausiid Euphausia crystal- lorophias but not in Euphausia superba. The composition of carotenoids was not investi- gated but had been analyzed by others (26-28). Fatty Acid Composition of Total Lipifls The composition of the fatty acids of total lipids of Euphausia superba is similar to that of other marine crustaceans and some marine fishes (29) (Tables 2 and 3). The main fatty TABLE 3 Branched Chain Fatty Acid Composition of Total Lipids of Euphausia superba Dana Sample 12/1977 3/1981 Ivl + ECL 13:0 i 14:0 i 15:0i 15:0 ai 16:0 i 17:0 i 17:0 br a 17:1 br 17:1 br 18:0i Phytanic b acid 228 12.6 tr. n.d. 242 13.6 0.05 ~+ 0.01 n.d. 256 14.6 0.19 (cid:12)9 0.00 0.31 -+ 0.15 256 14.7 0.21 -+ 0.01 0.24-+0.07 270 15.6 0.09-+ 0.03 0.10-+ 0.06 284 16.6 0.54-+ 0.05 0.20_+ 0.02 284 16.4 tr. 0.09 -+ 0.02 282 16.5 0.05 (cid:127) 0.03 0.11 -+ 0.08 282 16.2 tr. 0.10 -+ 0.05 298 17.6 tr. 0.10 -+ 0.01 326 17.7 2.82-+ 0.41 1.2 + 0.43 Data are expressed as wt % of total fatty acids and represent means +- standard deviation of 3 separate experiments. tr. = trace; n.d. = not detected; br. = branched; i = iso; ai = anteiso. apresumably 7-methylhexadecanoic acid. b3,7,11,15-tetramethylhexadecanoic acid. TABLE 2 Fatty Acid Composition of Total Lipids of Euphausia superba Dana Sample 12/1977 3/1981 M+a ECL b 10:0 186 10.0 11:0 200 11.0 12:0 214 12.0 13:0 228 13.0 14:0 242 14.0 14:1 240 13.8 15:0 256 I 5.0 15:1 254 14.8 16:0 270 16.0 16:1 (n-7) 268 15.7 16:1 (n-?) 268 15.8 16:2(n-6) 266 15.6 16:3 264 15.5 16:4(n-3) 262 15.4 17:0 284 17.0 17:1 282 16.7 17:1 282 16.8 18:0 298 18.0 18:1(n-7) 296 17.8 18:1(n-9) 296 17.7 18:l(n-?) 296 17.9 18:2(n-6). 294 17.6 18:3(n-3) 292 17.6 18:3(n-6) 292 17.3 tr. tr. tr. tr. 0.23 -+ 0.06 0.22 -+ 0.06 0.04 -+ 0.01 0.07 -+ 0.04 11.33 (cid:127) 1.48 15.23 (cid:127) 2.31 tr. 0.19 (cid:127) 0.01 0.34 + 0.01 0.27 -+ 0.05 tr. 0.04 -+ 0.03' 25.91 -+ 2.33 31.79 -+ 1.73 7.26 -+ 0.35 7.37 -+ 0.34 0.09 -+ 0.13 0.30 -+ 0.01 i 0.82 -+ 0.01 0.12 + 0.06 tr. 0.29 -+ 0.01 0.74 _+ 0.06 0.48 (cid:127) 0.14 0.06 _-L- 0.02 0.17 -+ 0.15 tr. 0.41 -+ 0.05 tr. 0.12 -+ 0.06 1.21 +_ 0.18 2.14 (cid:127) O.23 8.32 -+ 0.54 7.49 -+ 0.79 10.13 -+ 2.20 10.52 +- 0.90 tr. 0.09 -+ 0.05 1.58 + 0.09 0.74 -+ 0.38 0.47 _+ 0.02 0.33 -+ 0.07 0.21 (cid:127) 0.06 0.57 -+ 0.35 Sample 121977 3/1981 M+a ECL b 18:4(n-3) 290 17.4 0.67 -+ 0.07 0.62 -+ 0.49 19:0 312 19.0 tr. 0.11 -+ 0.16 19:1 310 18.8 0.12 -+ 0.04 0.20 -+ 0.09 19:2 308 18.7 tr. 0.07 _+ 0.05 20:0 326 20.0 0.04 -+ 0.00 0.19 -+ 0.14 20:1(n-7) 324 19.8 0.40 -+ 0.01 0.50 -+ 0.09 20:1(n-9) 324 19.7 0.77 -+ 0.04 1.35 _+ 0.23 20:2 322 19.6 tr. 0.08 -+ 0.06 20:4(n-3) 318 19.5 0.46 -+ 0.10 0.22 -+ 0.06 20:5(n-3) 316 19.3 12.71 -+ 1.57 7.83 +- 1.27 21:0 340 21.0 tr. tr. 21:5(n-3) 330 20.2 0.42 _+ 0.03 0.30 _+ 0.18 22:0 354 22.0 0.14 -+ 0.03 tr. 22:1(n-7) 352 21.6 0.29 -+ 0.17 0.41 +_ 0.16 22:1(n-9) 352 21.5 0.51 -+ 0.06 1.22 _+ 0.33 22:5(n-3) 344 21.2 0.54 -+ 0.09 0.24 -+ 0.11 22:5 344 21.4 tr. 0.04 + 0.03 22:6(n-3) 342 21.1 5.41 -+ 0.51 2.60 _+ 0.79 23:1 366 22.5 tr. 0.11 -+ 0.07 24:0 382 24.0 tr. tr. 24:1 380 23.6 tr. 0.15 + 0.11 25:0 396 25.0 tr. tr. Others c - - 3.95 2.45 "Data are expressed as wt % of total fatty acids and represent means -+ standard deviation of 3 separate experi- ments. tr. = trace. aM+: molecular weight of fatty acid methyl ester as determined by GLC/MS. bECL: equivalent chain length, calculated by plotting chain length (as carbon number) versus retention time on CP SIL 5. Cpredominantly branched chain fatty acids as given in Table 3 in detail. LIPIDS, VOL. 19, NO. 11 (1984)
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`824 H. FRICKE, G. GERCKEN, W. SCHREIBER AND J. OEHLENSCHLAGER < .~, < ..t- < oo @I i I~~ '~ i I I~ ~l+i+I+l+i+~+ill I§ I I I l+i +~ i ~+I+t+I+I+I i i I+~ I I I I +i @ I ~ ~ § ~ ~ +1 l+i I I I I +I II~~ ~'~l~d I +1 ~ +1 § +1 +l I I +I I I I I +~ ~, 'R. +l +l ~ +~ +l +1 § ~~~.~ @ q) ~ ,m @ + < o +, e-, ~ (cid:12)9 ..~ m ~. r..) o LIPIDS, VOL. 19, NO. 11 (1984)
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`z U .., ,.d z >-, < -I- r r162 oD It'-- < L~ < - e'- r ANTARCTIC KRILL LIPIDS I ~ I ~ ~ i i I ~ ~ I ~o~ I I o', it-. .,~ Ir I I I i~ ~r I ,,...; t ~ ..,.2 r oo ~oo~-ooo- o~o oo- o + +1 + +l +1 +1 +1 +l +1 I +1 +1 +t I +1 +1 +r +1 I +l +l +1 ~ +t +l +1 I +1 I +1 § +l § +1 +l +1 l+l +l+l+l+l+l+l+lll+l+ll+ll+l +i +~ +I +~ +t +i +i +i +~ +I I L I +i +i +i I I I l I +i =~=~&&~&~'E~ ,A E E ii =~. .. c, r II 3i ~..- m 0 825 LIPIDS, VOL. 19, NO. I1 (1984)
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`826 H. FR1CKE, G. GERCKEN, W. SCHREIBER AND J. OEHLENSCHL.~GER acids are 14:0 (11-15%), 16:0 (26-32%). 16:1(n-7) (7%), 18:1(n-9) (10%), 18:1(n-7) (8%), 20:5(n-3) (8-13%) and 22:6(n-3) (3-5%). Odd-numbered fatty acids with chain lengths ranging from C-11 to C-25 also were found in trace amounts and verified by GLC/MS. In the unsaturated fatty acids the species of the (n-3) series are dominant, while (n-6) fatty acids are found only to a limited extent. This also has been reported for marine shrimps and fish (29- 31). Several branched-chain fatty acids ranging from C-13 to C-18 (straight-chain length) were found, most of them belonging to the iso- or anteiso series. Among the multi-branched-chain fatty acids phytanic acid (32-34), which was the main component, amounted up to 3% of total fatty acid content. The samples from the early season 1977 contain more unsaturated fatty acids, especially 20:5(n-3) and 22:6(n-3), and less saturated fatty acids such as 14:0 and 16:0 than the sample from March 1981. This difference in the fatty acid compositions seems to be a seasonal phenomenon which also was reported by Shibata (2). In most of the investigations of krilt lipids the fatty acids were determined only by their retention behavior (3,35). In this study it was possible to determine the mass, and hence the chain length and number of double bonds, for all fatty acids by the combination of GLC/MS. The number of 57 analyzed fatty acids exceeds that reported by Golovnya et al. (36), who used the same technique. According to their ECL values 20:1 and 22:1 belong to the (n-7)and (n-9) series and not to the (n-11) series (36). The data found suggest that a (n-7) monoene series is present carrying from 16:1 (n-7) through 18:1(n-7) and 20:1(n-7) to 22:1(n-7) (37,38). Arachidonic acid which was found by Clarke (3), Golovnya (36) and Bottino (5) in krill, and by Bottino in a shrimp (39) as a minor compo- nent, was not found. Dembitskii (40) showed that marine crustacea contained high levels of lipids with alkenyl side chains. In the samples investigated neither free aldehydes nor dimeth- ylacetals after derivatization could be detected. Short chain, medium chain and hydroxy fatty acids (~<C-12) were not detectable even after transesterification to the corresponding benzyl esters (19). Fatty Acid Composition of Lipid Classes The analysis of fatty acids of individual lipid classes indicates different fatty acid composi- tions for phospholipids (Table 4) and TG (Table 5). Fatty acids in TG are mostly satu- rated or monounsaturated with 14:0, 16:0, 16:1(n-7), 18:1(n-7) and 18:1(n-9) as dominat- ing species. Polyunsaturated fatty acids were found only in small amounts. In phospholipids phytanic acid was detected only in traces, but it represented 5.6% of TG fatty acids. The phospholipids and FFA have 16:0, 20:5(n-3) and 22:6(n-3) as principal fatty acids. In the individual lipid classes a difference can be seen between the December samples (1977) and the March samples (1981). The lipid classes of the December samples contain more saturated fatty acids and less unsaturated fatty acids than the March 1981 samples. The discrepancy in the seasonal changes of the fatty acid composition of total lipids as mentioned above and that of the individual lipid classes is caused by the different lipid class composition with varying relative amounts of TG. The positional analysis of the fatty acids in the main phospholipids PC and PE (Table 6) shows that saturated fatty acids are commonly linked to the sn-1 position and that the sn-2 position is preferred by unsaturated fatty acids. In this respect krill has the same fatty acid dis- tribution as other marine animals (41). TABLE 6 Fatty Acid Positional Analysis in Phosphatidylcholine (PC) and Phosphatidylethanolamine (PE) of Euphausia superba Dana (1977 Sample) Phospholipid PC PE sn-position sn- 1 an-2 sn- 1 sn- 2 14:0 3.5 2.3 0.7 0.8 16:0 60.8 4.7 45.4 3.3 16:1(n-7) 1.5 5.4 0.7 0.5 18:0 1.9 0.8 5.6 1.5 18:1(n-7) 11.1 tr. 24.0 0.9 18:1(n-9) 3.5 22.0 4.8 2.9 18:2(n-6) 0.6 4.8 0.6 0.6 20:5(n-3) 5.6 27.7 5.7 31.3 22:6(n-3) 2.1 11.1 3.5 41.3 Others 9.4 21.2 9.0 16.9 Data are expressed as wt % of fatty acids in one position from one experiment. TABLE 7 Composition of the Free Sterol Fraction in Euphausia superba Dana Sample 12/1977 3/1981 Cholesterol a 70.0 -+ 5.9 75.5 -+ 3.7 Desmosterol b 18.2 -+ 1.4 17.7 _+ 1.1 22-Dehydrocholesterol c 11.5 -+ 4.8 6.0 _+ 3.5 Others 0.8 +- 0.5 1.0 -+ 0.7 Total 100.5 100.2 aCholesta-5-en- 3/~-ol b Cholesta- 5,24-dien - 3fl-ol c 2 2-cis/trans-cholesta- 5,2 2-dien- 3t3-ol LIPIDS, VOL. 19, NO. 1! (1984)
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`ANTARCTIC KRILL LIPIDS 827 Sterols The krill samples contained 3 sterols as major components identified by GLC/MS and traces of other sterols and sterol esters with un- known structure. The proportions of choles- terol, desmosterol and 22-dehydrocholesterol are given in Table 7. Cholesterol, which cannot be synthesized de novo in marine crustaceans (42), is the main sterol. Desmosterol and 22- dehydrocholesterol levels are very high. These sterols are assumed to be intermediates in the conversion of dietary sterols to cholesterol (42, 43). A small amount of 22-dehydrocholesterol, but no desmosterol, also was detected in the Arctic euphausiid Meganyctiphanes norvegica, whereas the herbivorous copepod Calanus finnmarchicus contained 14.1% 22-dehydro- cholesterol and 27.7% desmosterol besides cholesterol as main sterol (44). ACKNOW LEDGMENTS Mrs. I. Wasum and Dr. W. K6nig did the GLC/MS analyses. This work was supported by the Federal Ministries of Food, Agriculture and Forestry and the Federal Ministries of Science and Technology. One of us, H.F., thanks the latter for financial support. REFERENCES 1. Grantham, G.J. (1977) Southern Ocean Fisheries Survey Progr. GLO/SO/77/2, FAO, Rome. 2. Shibata, N. (1983) Bull. Jap. Soc. Sci. Fish. 49, 259-264. 3. Clarke, A. (1980) J. Exp. Mar. Biol. Ecol. 43, 221-236. 4. Rzavskaja, F.M., Sakaeva, E.A., and Dubrovskaja, T.A. (1979) Rybnoe chozjajstvo 1979(10), 53-54. 5. Bottino, N.R. (1973) Fed. Proc. Fed. Am. Soc. Exper. Biol. 32, 562. 6. Bottino, N.R. (1974) Mar. Biol. 27, 197-204. 7. Bottino, N.R. (1975) Comp. Biochem. Physiol. 50 B, 479-484. 8. Pierce, R.W., van der Veen, J., and Olcott, H.S. (1969) J. Agr. Food Chem. 17, 367-369. 9. van der Veen, J., Medwadowski, B., and Olcott, H.S. (1971) Lipids 6,481-485. 10. Nonaka, J., and Koizumi, C. (1964) Bull. Jap. Soc. Sci. Fish. 30, 630-634. 11. EUingsen, T.E. (1982) Biokjemiske studier over antarktisk krill, Ph.D. Thesis, University of Trondheim, 239-316. 12. Mori, M., and Hikichi, S. (1976) The Report of the Central Res. Lab. of Nippon Suisan Co., Ltd. No. 11, 11-17. 13. Hempel, G., Sahrhage, D., Schreiber, W., and Steinberg, R. (1979) Arch. FischWiss. 30 (Beih. 1), 1-119. 14. Christians, O., Birnbaum, A., Leinemann, M., Manthey, M., and Oehlenschlffgar, J. (1982) Arch. FischWiss. 33 (Beih. 1), 143-170. 15. Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. 16. Kates, M. (1972) Techniques of Lipidology, North-Holland Publishing Company, Amsterdam. 17. Broekhuyse, R.M. (1968) Biochim. Biophys. Acta 152, 307-315. 18. Morrison, W.R., and Smith, L.M. (1964)J. Lipid Res. 5, 600-608. 19. Klemm, H.P., Hintze, U., and Gercken, G. (1973) J. Chromatogr. 75, 19-27. 20. Ballantine, J.A., Roberts, J.C., and Morris, R.J. (1980) J. Exp. Mar. Biol. Ecol. 47, 25-33. 21. Pugb, E.L., Kates, M., and Hanahan, D.J. (1977) J. Lipid Res. 18, 710-716. 22. Heckers, H., Dittmar, K., Melcher, F.W., and Kalinowski, H.D. (1977) J. Chromatogr. 135, 93-107. 23. Fricke, H., and Schreiber, W. (1983) Naturwiss. 70, 308-309. 24. Ackman, R.G. (1982) in Proceedings of the Second International Conference on Aquaculture Nutrition: Biochemical and Physiological Ap- proaches to Shellfish Nutrition (Pruder, G.D., Langdon, C.J., and Conklin, D.E., ed.) Baton Rouge, Louisiana, pp. 358-376. 25. Kubota, K. (1980) J. Jap. Soc. Food Nutr. 33, 191-193. 26. Czeczuga, B., and K4yszejko, B. (1978) Pol. Arch. Hydrobiol. 25,657-662. 27. Czerpak, R., Jackowska, H., and Mical, A. (1980) Pol. Polar Res. 1, 139-145. 28. Yamaguchi, K., Miki, W., Toriu, N., Kondo, Y., Murakami, M., Konosu, S., Satake, M., and Fujita, T. (1983) Bull. Jap. Soc. Sci. Fish. 49, 1411-1415. 29. Ackman, R.G. (1980) in Advances in Fish Sci- ence and Technology (Connell, J.J., ed.) pp. 86- 103, Fishing News Books Ltd., Farnham, Surrey, U.K. 30. Chanmugam, P., Donovan, J., Wheeler, D.J., and Hwang, D.H. (1983) J. Food Sci. 48, 1440-1441 and 1462. 31. Bell, M.V., Simpson, C.M.F., and Sargent, J.R. (1983) Lipids 18, 720-726. 32. Hansen, R.P. (1969) Aust. J. Sci. 32, 160-161. 33. Hansen, R.P., and Meiklen, S.M. (1970) J. Sci. Fd. Agric. 21,203-206. 34. Ackman, R.G. (1968) Comp. Biochem. Physiol. 24, 549-565. 35. Ackman, R.G. (1970) J. Fish. Res. Bd. Canada 27, 513-533. 36. Golovnya, R.V., Kuzmenko, T.E., Samusenko, A.L., and Grigoreva; D.N. (1981)Appl. Biochem. Microbiol. 17, 47-53. 37. Ratnayake, W.N., and Ackman, R.G. (1979) Lipids 14, 795-803. 38. Ratnayake, W.N., and Ackman, R.G. (1979) Lipids 14, 804-810. 39. Lilly, M.L., and Bottino, N.R. (1981) Lipids 16, 871-875. 40. Dembitskii, V.M. (1979) Sov. J. Mar. Biol. 5(5), 86-91. 41. Brockerhoff, H., Yurkowski, M., Hoyle, R.J., and Ackman, R.G. (1964) J. Fish. Res. Bd. Canada 21, 1379-1384. 42. Morris, R.J., and Culkin, F. (1977) Oceanogr. Mar. Biol. Ann. Rev. 15, 73-102. 43. Gordon, T. (1982) J. Am. Oil Chem. Soc. 59, 536-545. 44. Sargent, J.R., and Falk-Petersen, S. (1981) Mar. Biol. 62, 131-137. Received April 30, 1984 LIPIDS, VOL. 19, NO. 11 (1984)
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