The carotenoids of eggs of wild and farmed cod (Gadus morhua)

Camp, Biochem, Physiol.Vol. 106B,No. 2, pp. 237-242, 1993 Printed in Great Britain

0305-0491/93 $6.00+ 0.00 © 1993Pergamon Press Ltd

THE CAROTENOIDS OF EGGS OF WILD A N D F A R M E D COD (GADUS MORHUA) MERETE GRUNG, YNGVAR STAVSETSVENDSEN* and SYNNOVELIAAEN-JENSEN Organic Chemistry Laboratories, Norwegian Institute of Technology, University of Trondheim-NTH, N-7034 Trondheim-NTH, Norway (Fax 473 59-4256) (Received 25 February 1993; accepted 31 March 1993) Abstract--1. Eggs of wild cod, and of farmed cod fed (a) a diet supplemented with astaxanthin and (b) a diet supplemented with both astaxanthin and canthaxanthin, were analysed with respect to carotenoids. 2. The total carotenoid contents in eggs were 0.7 ppm for wild cod and 0.5 ppm for farmed cod. 3. Cod, having white flesh, deposit ketocarotenoids in the eggs, preferably astaxanthin. 4. Canthaxanthin can replace astaxanthin in the eggs, but astaxanthin appears to be deposited preferentially when both carotenoids are present in the diet. 5. The isomer distribution of (3S, YS):(3R, 3'S, meso):(3R, 3'R) astaxanthin in the eggs reflected the isomer composition of the diet. 6. Echinenone, 4'-hydroxyechinenone, adonixanthin and zeaxanthin encountered in cod eggs may represent reductive metabolites of canthaxanthin and astaxanthin.

INTRODUCTION

Cod is gaining increasing interest as a potential product in Norwegian aquaculture. In contrast to salmonids, where carotenoids account for the pinkish colour of the flesh, the flesh of the cod is normally white (Foss, 1985). However, the eggs of wild cod are slightly pinkish in colour. The ovaries of Pacific cod (Gadus morhua macrocephalus) have been investigated by Miki et al. (1982). The carotenoids in the skin of cod (Gadus morhua) have previously been studied (Foss, 1985). Tunaxanthin (1) was the main carotenoid, and astaxanthin (2) accounted for 10-31% of the total carotenoid. Isomer analysis of astaxanthin (2) revealed the dominance of the 3R, YR-isomer (56% of total). This unexpected isomer distribution was explained by the cod feeding on the crab Cancer pagarus. Two other carotenoids, tentatively 3,4,3'-trihydroxy-fl,fl-carotene-4-one (3) and fl,fl-carotene-3,4,3'4'-tetrol (4), were also detected. The purpose of the present study was (i) a comparative investigation of the carotenoid composition of eggs of wild and farmed cod, and (ii) studies on the carotenoid composition of eggs in farmed cod after supplementing the diet with astaxanthin and canthaxanthin.

MATERIALS AND METHODS

Biological material Wild cod eggs. Maturing ovaries (cod roe) were taken from killed cod, which were caught off the *Finnmarksforskning, Pb. 476, N-9601 Hammerfest, Norway.

coast of Finnmark, North Norway, in April 1992. The eggs were kept frozen at - 2 5 ° C . Farmed cod. The cod were captured alive in 1989 and kept in nets at Finnmark Research Centre, Hammerfest, North Norway on the basic diet containing astaxanthin (2) for 2 years. Canthaxanthin (7, Carophyll Red, 10%, Roche) was added to the basic diet of one net from 15 November 1991 to 20 February 1992 when food intake ceased and the spawning season started. In March, 60 cod (average weight: 6 kg; age more than 5 years) were transferred from nets to a spawning enclosure, where spawning took place until the end of April. The feed composition of the basic diet, including astaxanthin (2), is given in Table 1. The canthaxanthin-supplemented diet contained, in addition, Carophyll Red (10%, Roche) 0.1% (providing canthaxanthin, 100mg/kg diet). The Carophyll Red 0 0 % ) was dissolved in water and dispersed in the feed during mixing of the ingredients. The feed was kept frozen at -25°C. Farmed cod eggs. Natural spawned eggs (age: 1-3 days, fertilized) were collected 6 April 1992 from the spawning enclosure. The eggs were kept frozen at -25°C. Methods The eggs were ground in a mortar, and the carotenaids extracted with acetone. After concentration under reduced pressure, the carotenoids were transferred to diethyl ether by addition of water. The aqueous phase was extracted repeatedly with ether to effect complete transfer of the carotenoids. The combined ethereal layer was washed with water and dehydrated over anhydrous sodium sulphate. The solvent was evaporated under reduced pressure. 237

238

MERETE GRUNG et aL Table 1. Feed composition of the basic diet Basic diet

% weight

Herring meal (Nor Sea Mink, Tess) Salmomix 45% (Tess) (75 mg/kg astaxanthin, Carophyll Pink) Fish muscle Squid Vitamin concentrate Capelin oil (containing astaxanthin ca 20 mg/kg)

Colourless contaminants were removed by filtration from cold acetone and by TLC or CC on silica using hexane as eluent. The carotenoid content was determined by VISspectroscopy using El~m 1,/o = 2000 at 2m~ in acetone. Separation of individual carotenoids was effected by TLC using mixtures of acetone and hexane as eluents. Chemical derivatization. Astaxanthin (2) was esterified by dissolving 2 (ca 0.1 mg) in dry pyridine (0.5-1.0 ml) and adding (-)-camphanic acid chloride (30-90 mg) at 0°C. The reaction was monitored by TLC (silica, diethyl ether as eluent). After the reaction was completed, a saturated aqueous solution of sodium chloride was added. The astaxanthin (2) camphanates were extracted with diethyl ether, washed repeatedly with water and purified by TLC prior to HPLC analysis. H P L C conditions. Analysis of the extract (Grung and Liaaen-Jensen, 1992): Brownlee Spheri-5 silica 5-micron column 220 x 4.6mm, gradient elution: linear increase 0-30% acetone in hexane for 30 min, thereafter stationary conditions (30% acetone in hexane) for 15 min, flow 1.25 ml/min. The detection wavelength was 450nm and VIS spectra were recorded in the eluent during the chromatographic run. Both a normal silica column, and a silica column conditioned with orthophosphoric acid (Vecchi et al., 1987) in order to elute the ~-ketols without tailing, were used. Analysis of astaxanthin (2) dicamphanates (Vecchi and Miiller, 1979): Brownlee Spheri-5 cyano 5 micron column 220 x 4.6 mm, eluent was hexane:isopropyl acetate: acetone: methanol 76:17: 7: 0.1. The flow was 1.5 ml/min and the detection wavelength was 491 nm. Individual carotenoids. Echinenone (g): R T 9.09 rain, inseparable from synthetic 8 by HPLC, VIS 2m~ nm 458 (HPLC-eluent). Canthaxanthin (7): R r 14.16 rain, inseparable from synthetic 7 by HPLC, VIS Area~nm 469 (HPLC-eluent), MS m/z 564 (M, 100%), 562 (M-2, 10%), 548 (M-16, 15%), 546 (M-18, 17%), 532 (M-16-16, 27%), 472 (M-92, 19%). 4'-Hydroxyeehinenone (5): RT 17.05 min, less polar than authentic Y-hydroxyechinenone (ex Botryococcus braunii) by HPLC, VIS 2m~xnm 458 (HPLCeluent). Astaxanthin (2): RT 18.70min, inseparable from synthetic 5 by HPLC, VIS 2m~xnm 474 (HPLC-eluent), MS m/z 596 (M). Synthetic astaxanthin (2) was esterified with ( - ) camphanic acid chloride and analysed by HPLC

22.0 23.9 45.0 7.5 1.0 0.5

(Vecchi and Miiller, 1979). The result is given in Table 2. Astaxanthin (2) from the feed, from eggs of wild cod, eggs of farmed cod fed the basic diet and eggs of farmed cod fed the basic diet supplemented with canthaxanthin (7) was esterified with ( - )-camphanic acid chloride and analysed by HPLC; for results see Table 4. Rr values varied somewhat for each analysis, but the identification was demonstrated in each case by co-chromatography with the authentic mixture of diastereomeric dicamphanates. Adonixanthin (10): R r 20.46 min, inseparable from synthetic 10 by HPLC, VIS 2max n m 460 (HPLCeluent). Lutein (6): R v 22.22 min, inseparable from authentic 6 (ex. B. braunii) by HPLC, VIS 2m~ nm 445, 473, % III/II (Ke et al., 1970)= 69 (HPLC-eluent). Zeaxanthin (9): RT 22.42min, inseparable from synthetic 9 by HPLC, VIS 2m~xnm 451,479, % III/II = 39 (HPLC-eluent). Fucoxanthin (12)-like: R x 27.10, inseparable from authentic fucoxanthin (12) (ex. Fucus serratus) by HPLC, VIS Area~ nm 447, 467, % III/II = 0 (HPLCeluent). Unidentified: eggs of wild cod contained three unidentified carotenoids (6 + 6 + 8% of total) less polar than astaxanthin (2) with 2m~x476 nm (HPLCeluent). Eggs of cod fed the basic diet had four minor unidentified carotenoids (2 + 2 + 2 + 3% of total) whereas eggs of cod fed the canthaxanthin (7) supplemented diet had four unidentified carotenoids (2 + 2 + 2 + 1% of total). RESULTS AND DISCUSSION

Carotenoids in the feed Basic feed. Carotenoid content: the basic feed was analysed. An exact measurement of the carotenoid content was difficult because of the high amount of lipid. After extraction with acetone and chromatography on silica, the carotenoid content was determined by VIS spectroscopy to 1>20 ppm. Table 2. Retention times and % composition of dicamphanates of astaxanthin (2) of synthetic origin RT 6.78 7.37 8.01 8.62 9.36 10.21

%

Isomer 2"~ 21 f 23 5"~_ 47 f 52 2"~ 23 f 25

cis-(3R, YR)-astaxanthin (2¢) (3R, YR)-astaxanthin (2¢) cis-(3R, YS, meso)-astaxanthin (2b) (3R, YS, meso)-astaxanthin (2b) cis-(3S, YS)-astaxanthin (2a) (3S, YS)-astaxanthin (2a)

239

Carotenoids of cod eggs Table 3. Carotenoidsin the basic feed and three differenttypes of cod eggs % of total carotenoids Carotenoid Basic feed Wild cod Cod fed 2 Cod fed 2 and 7 Echinenone (8) Canthaxanthin (7)

4'-Hydroxyechinenone(5) Astaxanthin(2) Adonixanthin(lfi) Lutein(6) Zeaxanthin(9) Fucoxanthin(12)-like Unidentified(sum)

3

3 93

76 4

traces 1 2

20

Individual carotenoids: HPLC-analysis (Grung and Liaaen-Jensen, 1992) revealed that astaxanthin (2) was the main carotenoid (93% of total). Identification of the other carotenoids was difficult due to small amounts and contamination of lipids. RT values a n d on-line VIS data indicated the presence of 4'-hydroxyechinenone (5) and lutein (6). The carotenoid composition according to HPLC data is given in Table 3. Isomer distribution of astaxanthin (2): the isomer distribution of astaxanthin (2) was analysed by the method of Vecchi and Mfiller (1979), and is given in Table 4. Astaxanthin (2) was esterified with ( - ) camphanic acid chloride and the resulting diastereomers analysed by HPLC. The pigment source of the basic feed consisted of Salmomix (containing synthetic 2) and ground farmed salmonids (also containing 2 of synthetic origin). The results (3S, 3'S (2a): meso (2b): 3R, YR (2c) = 23:50:27) are in accordance with synthetic astaxanthin (2) (1:2:1) being the source of astaxanthin (2) in the feed. Canthaxanthin-supplemented feed: the feed supplemented with canthaxanthin contained Carophyll Red (canthaxanthin (7), 10%) 1 kg/(1000 kg feed); that is 100 ppm canthaxanthin (7) in addition to the 20 ppm astaxanthin (2) in the basic feed. Carotenoids in the eggs

Eggs from three different cod sources were analysed with respect to carotenoids: (a) wild cod; (b) cod fed a diet containing astaxanthin (2) (ca 20 ppm); and (c) cod fed a diet containing both astaxanthin (2) (ca 20 ppm) and canthaxanthin (7) (100 ppm). Carotenoid content. The eggs of cod were slightly pinkish in colour. Eggs of wild cod contained 0.7 ppm carotenoids of the wet weight. The carotenoid content of eggs from farmed cod was 0.5 ppm, irrespec-

32 5 39 6 1 1 6 7

78 8 1 1 3 9

tive whether the carotenoid source was astaxanthin (2) or both astaxanthin (2) and canthaxanthin (7). Eggs of salmonids, which have a pinkish colour, have been examined for carotenoids by several authors (Bjerkeng et al., 1991; Craik, 1985; Craik and Harvey, 1986). The carotenoid concentration is commonly between 3 and 12 ppm. Miki et al. 0982) have analysed the ovaries of Pacific cod (Gadus morhua macrocephalus). The colour of the ovaries appeared orange-brown, and the concentration of carotenoids was 4 ppm of the wet weight. This high concentration may reflect that the eggs were newly fertilized, or the fact that this is a different subspecies of cod. Individual carotenoids. The individual carotenoids encountered in eggs of different origin, identified after HPLC, are listed in Table 3. The carotenoid content of cod eggs was low, which made the identification of the individual carotenoids difficult. The major carotenoids, astaxanthin (2) and canthaxanthin (7) were identified by Rr, on-line VIS spectra, co-chromatography by HPLC with synthetic references and MS. Astaxanthin (2) was esterified by the camphanate method (Vecchi and Mfiller, 1979), and the diastereomeric mixture of dicamphanates of (2a) (2b) and (2e) astaxanthin was inseparable from that of an authentic mixture of these three dicamphanates. Echinenone (8), lutein (6) and zeaxanthin (9) were only characterized by RT, VIS-spectra and co-chromatography with authentic substances by HPLC. These identification criteria do not meet the minimum requirement for the unequivocal identification of a carotenoid (Liaaen-Jensen, 1978). However, the probability of finding such carotenoids in cod eggs is high. The separation capacity of the HPLC-system in this polarity region is good, and this system is capable of separating lutein (6) and zeaxanthin (9). The VIS-spectra and spectral fine-structure (Ke et al., 1970) were as expected for 6, 8 and 9 respectively. The identification of adonixanthin (10) was based on RT, VIS-spectra and co-chromatography with

Table 4. Isomerdistributionof astaxanthin (2) in the feed and in three differenttypes of cod eggs % of total astaxanthin (2) Astaxanthin (2) isomer

3S, 3'S (2a) 3R, 3"S, (meso)(2b) 3R, YR (2¢)

Feed 23 50 27

Wild cod 50 34 16

Cod fed 2 34 48 18

Cod fed 2 and 7 34 50 16

HO''~

~

~

(3R3'R)~n(2c)

(3S, 3'R )-Astaxanthin (2b)

Scheme 1

"

O ~OH

O ,~OH

O

H o

~

B

v

O

OH

OH

3,4,3'-Trihydroxy-~,~-carotene 4-one (3)

O

OH OH

Fucoxanthin (12)

3'-Hydroxyechinenone (11)

~ ~ ~~~ ~ ~ ~ 1 ~ )

OH

C Z

Carotenoids of cod eggs synthetic 10 by HPLC. The fact that this carotenoid exhibited tailing on a normal silica HPLC-column, and no tailing on a modified silica column (Vecchi et al., 1987) is compatible with an ~t-ketol. The carotenoid with R x 17.05 min was tentatively identified as 4'-hydroxyechinenone (5). Thus 2m~xat 458 nm indicated one keto group in conjugation with the fl,fl-carotene chromophore. Both the Rx value and the fact that the carotenoid exhibited no tailing on a normal silica column disfavoured an ~t-ketol. Upon co-injection with Y-hydroxyechinenone (11) was 5 less polar. The carotenoid with Rx 27.10 min had a polarity in accordance with a triol. Lutein (6)- and zeaxanthin (9)-like triols would exhibit spectral fine-structure % III/II (Ke et al., 1970) of 10 or 60%, and 2max of either 445 or 452 nm, respectively. The 2m~x at 447 and absence of spectral fine-structure indicated a fucoxanthin-like carotenoid, compatible with co-chromatography tests with fucoxanthin by HPLC. Echinenone (8), 4'-hydroxyechinenone (5), adonixanthin (10) and zeaxanthin (9) may represent reductive metabolites of canthaxanthin (7) and astaxanthin (2) (Schiedt et aL, 1986, 1988). The results compiled in Table 3 demonstrate that astaxanthin (2) is the major carotenoid in the eggs of wild cod and in farmed cod fed an astaxanthin (2)-containing diet. When the astaxanthin (2)-containing diet was supplemented with a 5-fold quantity of canthaxanthin (7) for three months prior to spawning, astaxanthin (2) was still slightly superior in the relative quantity encountered in the eggs. The result demonstrates the ability of cod to deposit canthaxanthin (7) in the eggs, and indicates a preference for astaxanthin (2) when both 2 and 7 were offered together. The total carotenoid content of the eggs was somewhat higher for wild cod than for farmed cod. Isomer distribution o f astaxanthin (2). The isomer distribution of astaxanthin (2) was determined for the three types of eggs of different origin, and is given in Table 4. Astaxanthin (2) was esterified with ( - ) camphanic acid chloride and the resulting diastereomers analysed by HPLC (Vecchi and Miiller, 1979). It is known that salmonids do not epimerize astaxanthin (2) resorbed from the feed (Storebakken et al., 1985). However, a slightly selective deposition of (3R, Y S , meso) (2b) and (3S, YS) (2a) has been observed in the skin of rainbow trout (Bjerkeng et al., 1992). The isomer distribution of the eggs of salmonids has been used to recognize escaped farmed salmonids in Norwegian rivers (Lura and Saegrov, 1991a, b). Since wild cod and farmed cod had different isomeric ratios for astaxanthin (2), it may be assumed that, for the cod too, the isomer distribution of astaxanthin (2) is determined by the ratio in the diet. Wild cod is likely to obtain its astaxanthin (2) from small crustaceans. Foss (1985) and Foss et al. (1987) have investigated the carotenoid composition and

241

isomeric distribution of astaxanthin (2) in some crustaceans. The ratio of 2a: 2b: 2c isomers of astaxanthin (2) in Artemia purpurea was (41:44:15) and in Calanus finmarchicus (83 : 3:14). A wild cod feeding on both A. purpurea and C. finmarchicus might achieve the isomeric composition of 2 encountered in the eggs. Farmed cod was expected to reflect the isomeric ratio 25:50:25 of 2a, 2b and 2e astaxanthin (2) present in the feed. However, the observed isomeric distribution of astaxanthin (2) suggests that the cod, kept in nets, also had access to crustaceans providing, particularly, (3S, 3'S)-astaxanthin (2a) from the sea. Acknowledgement--MG held a fellowship from the Norwegian Research Council of Science and Humanities. REFERENCES

Bjerkeng B., Storebakken T. and Liaaen-Jensen S. (1991) Response to carotenoids by rainbow trout in the sea: resorption and metabolism of dietary astaxanthin and canthaxanthin. Aquaculture 91, 153-162. Bjerkeng B., Storebakken T. and Liaaen-Jensen S. (1992) Pigmentation of rainbow trout from start feeding to sexual maturation. Aquaculture 108, 333-346. Craik J. C. A. (1985) Egg quality and egg pigment content in salmonid fishes. Aquaculture 47, 61-88. Craik J. C. A. and Harvey S. M. (1986) The carotenoids of eggs of wild and farmed Atlantic salmon, and their changes during development to the start of feeding. J. Fish Biol. 29, 549-565. Foss P. (1985) Carotenoids in aquatic animals. In Applied Carotenoid Chemistry--Algal Chemosystematics and Food Chain Studies, pp. 60-72. Ph.D. thesis, Norwegian Institute of Technology, Trondheim, Norway. Foss P., Renstr~m B. and Liaaen-Jensen S. (1987) Natural occurrence of enantiomeric and meso astaxanthin 7. Crustaceans including zooplankton. Comp. Biochem. Physiol. 86B, 313-314. Grung M. and Liaaen-Jensen S. (1992) Normal phase gradient HPLC separation of algal carotenoids and microalgal origin of carotenoids in coral eggs. In Marine Natural Products. Symposium Proceedings: 7th International Symposium on Marine Natural Products, p. 25. Capri. Ke B., Imsgard F., Kjosen H. and Liaaen-Jensen S. (1970) Electronic spectra of carotenoids at 77°K. Biochim. biophys. Acta 210, 139-152. Liaaen-Jensen S. (1978) Marine carotenoids. In Marine Natural Products. Chemical and Biological Perspectives (Edited by Scheuer P.), Vol. 2, pp. 1-73. Academic Press, New York. Lura H. and S~egrov H. (1991a) A method of separating offspring from farmed and wild Atlantic salmon (Salmo salar) based on different ratios of optical isomers of astaxanthin. Can. J. Fish. Aquat. Sci. 48, 429-433. Lura H. and S~egrovH. (1991b) Documentation of successful spawning of escaped farmed female Atlantic salmon, Salmo salar, in Norwegian rivers. Aquaculture 98, 151-159. Miki W., Yamaguchi K. and Konosu S. (1982) Comparison of carotenoids in the ovaries of marine fish and shellfish. Comp. Biochem. Physiol. 71B, 7-11. Schiedt K., Vecchi M. and Glinz E. (1986) Astaxanthin and its metabolites in wild rainbow trout (Salmo gairdneri R.). Comp. Biochem. Physiol. 83B, 9-12. Schiedt K. Vecchi M., Glinz E. and Storebakken T. (1988) Metabolism of carotenoids in salmonids. Metabolism of astaxanthin and canthaxanthin in the skin of

242

MERETE GRUNG et al.

Atlantic salmon (Salmo salar L.). Helv. Chim. Acta 71, 887-896. Storebakken T., Foss P., Austreng E. and Liaaen-Jensen S. (1985) Carotenoids in diets for salmonids--II. Epimerization studies with astaxanthin in Atlantic salmon. Aquaculture 44, 259-269. Vecchi M. and Miiller R. K. (1979) Separation of (3S, YS)-,

(3R, 3'R)- and (3S, 3'R)-astaxanthin via ( - )-camphanic acid esters. J. High Resol. Chromat. Chrom. Comm. 2, 195-196. Vecchi M., Glinz E., Meduna V. and Schiedt K. (1987) HPLC separation and determination of astacene, semiastacene, astaxanthin and other keto-carotenoids. J. High Resol. Chromat. Chrom. Comm. 10, 348-351.