Comp. Biochem. Physiol. Vol. 8611, No. 2, pp. 313-314, 1987 0305-0491/87 $3.00+0.00 Printed in Great Britain Pergamon Journals Ltd NATURAL OCCURRENCE OF ENANTIOMERIC AND MESO ASTAXANTHIN 7*-CRUSTACEANS INCLUDING ZOOPLANKTON PER Foss, BRITTA RENSTROM and SYNNOVE LIAAEN-JENSEN Organic Chemistry Laboratories, Norwegian Institute of Technology, University of Trondheim, N-7034 Trondheim-NTH, Norway (Received 14 February 1986) Abstract--1. The isomeric ratio of enantiomeric and meso astaxanthin (free and esterified) in the crustaceans Calanus finmarchieus, Euphausia superba, Thysanoessa inermis, Acanthephyra purpurea and Cancer pagurus is reported. 2. The ratios observed in C.finmarchicus and T. inermis, both considered as important feed ingredients for wild salmon, are compatible with those reported by others for wild salmon and with recent evidence demonstrating that salmonids obtain the three optical isomers of astaxanthin from the diet. 3. The origin of the three optical isomers of astaxanthin in zooplankton is discussed briefly. INTRODUCTION Previously we have reported the ratio of the natural occurrence of the three optical isomers 3S,3'S (1, Scheme 1), 3R,3'S (2, meso) and 3R, YR (3) of astaxanthin and its esters in lobster (Hommarus gam- marus; Ronneberg et al., 1980; Renstrom et al., 1982), shrimp (Pandalus borealis; Renstrom et al., 1981a) and flowers of Adonis annua (Renstrom and Liaaen- Jensen, 1981b). Schiedt et al. (1981) studied this ratio for wild salmon, which was remarkably similar for different species and geographical localities. Subsequent feed- ing experiments with synthetic, individual optical isomers of astaxanthin (1, 2 and 3) to rainbow trout and to salmon have demonstrated that salmonids resorb each isomer to an equal extent, are not able to carry out an epimerization at C-3,Y in astaxanthin, and that the isomeric ratio observed merely reflects the configuration of the astaxanthin present in the diet (Foss et al., 1984; Storebakken et al., 1985). A similar conclusion was also reached from studies on the isomeric ratio (1:2:3) of astaxanthin in zoo- plankton and salmonids in two Norwegian subalpine lakes (Storebakken et al., 1984). The ratio of the optical isomers of astaxanthin may be useful in food chain studies. MATERIALS AND METHODS Biological materials Euphausia superba, caught December 1978 near the Bouvet island, Thysanoessa inermis caught February 1982 near Svartnes, Balsf~ord, Norway, Calanus finmarchicus ® ® caught in May 1984 near Sunndalsora, Norway and Acan- thephyra purpurea caught May 1981, were stored frozen. Cancer pagurus was purchased at the local fish market 1980. Physical and chemical methods Isolation of the carotenoids. The carotenoids were extrac- ted with acetone at room temperature. Whole frozen ani- mals were extracted, except for C. pagurus where only the shell was used. Chromatography was carried out by TLC (SiO v 30% acetone-hexane). An extinction coefficient of El~m = 2100 was used, calculating the esters as astaxanthin equivalents. Astaxanthin diester. RE=0.70, inseparable from an authentic sample ex Pandalus borealis; VIS2~a~nm (acetone) 470, Astaxanthin monoester. R e = 0.46, inseparable from an authentic standard ex P. borealis; VIS ).max nm (acetone) 470. Astaxanthin. R E = 0.30, inseparable from synthetic as- taxanthin; VIS 2m~ x nm (acetone) 470; MS (200°C) m/z 596 (M +, 10%), 594 (M-2, 14%), 504 (M-92, 4%), 490 (M-106, 8%), 91 (100%). Astaxanthin dicamphanate. The mono- and diesters were submitted to anaerobic hydrolysis to astaxanthin as de- scribed elsewhere (Renstrom et al., 1981c). Astaxanthin (0.I mg) was esterified with (-)-camphanoyl chloride (30 90 rag), providing the dicamphanate R e = 0.84 (SiO2, diethyl ether); VIS '~max nm (acetone) 470 (Schiedt et al., 1981; Foss et al., 1984). The diastereomeric camphanates were submitted to HPLC by the standard procedure (Vecchi and Miiller, 1979). RESULTS AND DISCUSSION We now report the isomeric ratio (1:2:3) of five species of crustaceans: Thysanoessa inermis, Acan- ® 1 (3S, 3'S) A-P-A 2 (3R,3S; meso) B-P-A 3 (3R,3Ft) B-P-B 313
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`314 PEa. FOSS et al. Table 1. Carotenoid composition in per cent of total carotenoids, and ratio of optical isomers S,S : R,S (meso): R,R of free and esterified astaxanthin in some Crustaceans Cancer pagurus Carotenoid Euphausia superba Thysanoessa inermis Calanus finrnarchicus Acanthephyra purpurea shell ) 651 ~ 46) 43 22 Astaxanthin diester 64 (50 : 24: 26) (84: 5:11) Astaxanthin monoester 31 (9:21:70) 43 37 13 Astaxanthin 5 4 (55:7:38) 11 (83:3:14) 20 (20:44:15) 58 (20:24:56) thephyra purpurea, Cancer pagurus and the zoo- plankton Calanus finmarchicus, all from the North Sea and Euphausia superba from Antarctic waters, Table 1. Consistent with previous studies (Barbier et al., 1966; Batham et al., 1951; Fischer et al., 1955; Herring, 1973; Lenel et al., 1978; Wieser, 1965) free and esterified astaxanthin were the only carotenoids present. A mixture of the three astaxanthin isomers was demonstrated in each case. In E. superba and C. pagurus the R-configuration was dominant, whereas in T. inermis and C. finmarchicus the S-configuration dominated. The two species T. inermis and C. finmarchicus are recognized as important feed ingre- dients for wild salmon, and the ratios in these two crustaceans are compatible with the isomeric as- taxanthin mixture encountered in wild salmon (Schiedt et al., 1981). We have recently demonstrated that in the fresh- water zooplankton Daphnia magna optically pure (3S,3'S)-astaxanthin (I) was formed from optically pure (3R,3'R)-zeaxanthin (fl,fl-carotene-3,3'-diol) of algal origin (Partali et al., 1986). However, the origin of the R-configurated astaxanthin isomers (2 and 3) in zooplankton is still obscure. The formation of particularly 3 and 2 by zooplankton, presumably from phytoplankton carotenoid precursors will be studied. Acknowledgements--We thank Dr P. J. Herring, Institute of Oceanographic Sciences, Surrey, England, for the sample of Acanthephyra purpurea, cand. agric. Trond Storebakken, Research Station for Salmonids, Sunndalsora, Norway, for the sample of Calanusfinmarchicus and Dr Trond Ellingsen and Siv.ing. Olav S~eter, Institute of Biotechnology, this University for samples of Euphausia superba and Thy- sanoessa inermis, respectively. REFERENCES Barbier M., Charniaux-Cotton H. et Fried-Montaufier M. (1966) Presence dans les secondes antennes des males Talitrus saltator et Orchestia gammarella (Crustacea Am- phipodes) d'un taux relativement eleve d'astaxanthine. C. r. Acad. Sci. Paris, 263, 1508-1510. Batham E., Fisher L. R., Henry K. M, Kon S. K. and Thompson S. Y. (1951) Preformed vitamin A in marine crustacea. Biochem. J. 48. Fisher L. R, Kon S. K. and Thompson S. Y. (1955) Vitamin A and carotenoids in certain invertebrates III. Euphausiacea. J. mar. Biol. Assoc., UK 34, 81-100. Foss P. (1985) Applied carotenoid chemistry. Algal chemo- systematics and food chain studies. Dr. ing. Thesis, Univ. Trondheim-NTH, 327 p. Foss P., Storebakken T., Schiedt K., Liaaen-Jensen S., Austreng E. and Streiff K. (1984) Carotenoids in diets for salmonids I. Pigmentation of rainbow trout with the individual optical isomers of astaxanthin in comparison with canthaxanthin. Aquaculture 41, 213 226. Herring P. J. (I 973) The distribution of carotenoid pigments and lipids of some oceanic animals. 2. Decapod crus- taceans. J. mar. Biol. Assoc., UK 53, 339-568. Lenel R., Negre-Sadargues G. et Castillo R. (1978) Les Pigments Carotenoides Chez Les Crustaces. Arch. Zool. Exp. 119, 297-334. Partali V., Olsen Y., Foss P. and Liaaen-Jensen S. (1985) Carotenoids in food Chain Studies. 1. Zooplankton (Daphnia magna) response to a unialgal (Scenedesmus acutus) carotenoid diet, to spinach and to yeast diets supplemented with individual carotenoids. Comp. Bio- chem. Physiol. 82B, 767-772. Renstr~m B., Borch G. and Liaaen-Jensen S. (1981a) Nat- ural occurrence of enantiomeric and meso-astaxanthin. 4. Ex shrimps (Pandalus borealis ). Comp. Biochem. Physiol. 69B, 621-624. Renstr~m B. and Liaaen-Jensen S~ (1981b) Natural occur- rence of enantiomeric and meso-astaxanthin. 6. Esterified, optically pure (3S, YS)-astaxanthin from flowers of Ado- nis annua. Biochem. Syst. Ecol. 9, 249 250. Renstr~m B., Borch G., Skulberg O. and Liaaen-Jensen S. (1981c) Natural occurrence of enantiomeric and meso- astaxanthin. 3. Optical purity of (3S, YS)-astaxanthin ex Haematococcus pluvialis (green algae). Phytochemistry 20, 2561-2564. Renstrom B., Ronneberg H., Borch G. and Liaaen-Jensen S. (1982) Animal carotenoid 27. Further studies on the carotenoproteins crustacyanin and ovoverdin. Comp. Biochem. Physiol. 71B, 249-252. Ronneberg H., Renstrom B., Aareskjold K., Liaaen-Jensen S., Vecchi M., Leuenberger F. J., Miiller R. K. and Mayer H. (1980) Natural occurrence of enantiomeric and meso- astaxanthin. 1. Ex lobster eggs (Homarus gammarus). Helv. chim. Acta 63, 711 715. Schiedt K., Leuenberger F. J. and Vecchi M. (1981) Natural occurrence of enantiomeric and meso-astaxanthin. 5. Ex wild salmon ( Salmo salar and Oncorhynchus ). Heir. chim. Acta 64, 415-424. Storebakken T., Foss P., Asg~.rd T., Austreng E. and Liaaen-Jensen S. (1984) Carotenoids in food chain studies--Optical isomer composition of astaxanthin in crustaceans and fish from two sub-alpine lakes. Abstr. 7th Int. 1UPAC Symp. on Carotenoids, p. 3l. MiJchen 1984. Storebakken T., Foss P., Austreng E. and Liaaen-Jensen S. (1985) Carotenoids in diets for salmonids. II. Epimer- ization studies with astaxanthin in Atlantic salmon. Aquaculture 44, 259 269. Vecchi M. and MiJller R. K. (1979) Separation of (3S,3'S)-, (3R,3'R)-astaxanthin and (3S,3'R)-astaxanthin via (-)- camphanic acid esters. J. High Res. Chrom. 2, 195 196. Wieser W. (1965) Electrophoretic studies on blood proteins in an ecological series of isopod and amphipod species. J. mar. Biol. Assoc., UK 45, 507 523.
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