Metabolic pathways of carotenoids in chum salmon Oncorhynchus keta during spawning migration

Comp. Biochem. Physiol. Vol. 87B, No. 2, pp. 411416, 1987 Printed in Great Britain

0305-0491/87 $3.00+0.00 © 1987 Pergamon Journals Ltd

METABOLIC PATHWAYS OF CAROTENOIDS IN CHUM SALMON ONCORHYNCHUS KETA D U R I N G SPAWNING MIGRATION SEIICHI ANDO and MUTSUO HATANO Laboratory of Food Chemistry I, Faculty of Fisheries, Hokkaido University, Hakodate 041, Japan (Received 11 July 1986) Abstract--1. Based on the contents and individual composition of carotenoids in the muscle, serum and ovaries of chum salmon during spawning migration, the reductive metabolism of astaxanthin to zeaxanthin was presumed to take place in the muscle of both male and female. 2. The metabolic rates ofzeaxanthin and 4-keto-zeaxanthin in female serum were much faster than those in male serum.

INTRODUCTION Carotenoids are the main pigments of many aquatic animals and are of increasing importance for the pigmentation of cultured fish. In salmonids, carotenoids are responsible for the typical pink color of the muscle (Matsuno et al., 1980, 1984; Schiedt et al., 1981; Kitahara, 1983; Foss et al., 1984; Torrissen and Naevdal, 1984; Torrissen, 1985). Carotenoids are of dietary origin and are mainly deposited in the muscle. With sexual maturation, however, the carotenoids are mobilized from the muscle and transported to the integument and ovaries (Kitahara, 1983). Little has been reported concerning carrier proteins of carotenoids in the serum of salmon, although the muscle carotenoids are definitely transported into the utilization sites such as integument and ovaries via the serum. N a k a m u r a et al. (1985) have recently found that astaxanthin in the serum of the upstream migrating chum salmon is exclusively transported by high density lipoprotein. In previous papers (Ando et al., 1985, 1986a,b,c), we have demonstrated that various serum lipoproteins such as high density lipoproteins and very high density lipoproteins including vitellogenin transport the muscle astaxanthin into the integument and ovaries during spawning migration. During the studies on nuptial coloration of chum salmon we have noticed that the carotenoids-carrying lipoproteins have the ability to transport not only astaxanthin but also other carotenoids (Ando et al., 1985, 1986d). This might suggest that carotenoids have already been metabolized in the muscle. In the present study, we describe the possibility that astaxanthin is reductively converted to zeaxanthin in the muscle of chum salmon during spawning migration. MATERIALS AND METHODS Materials Specimens of chum salmon, Oncorhynchus keta, in different physiological states were used as the materials (Table 1). Blood was collected from the caudal vasculature of live salmon at each migration stage, and left at room temperature for several hours. The clotted blood was centrifuged to obtain the serum. 411

Extraction of carotenoids Carotenoids were extracted from muscle, serum and ovaries with acetone. The total carotenoid content was calculated, assuming the EI~~ value in acetone at 477 nm to be 2200. After concentration under reduced pressure, the carotenoids were transferred to diethyl ether by the addition of distilled water. The aqueous phase was extracted with ether several times. The combined ethereal layer was concentrated under reduced pressure. The residue was submitted to high performance liquid chromatography (HPLC). H P L C analysis of carotenoids HPLC was carried out on a Shimadzu LC-6A instrument with a Shimadzu SPD-2A VIS spectrophotometer set at 470 nm. The column used was a 250 x 4 mm I.D. stainless steel column packed with 5 pm Sumipax OA-2000 (Sumitomo Chemical Co.). Separation was achieved with a mobile phase of n-hexane-CH2C12-EtOH (50:20:0.5), flow-rate of 0.8 ml/min. Identification of each carotenoid was accomplished by co-thin layer chromatography and co-HPLC with authentic specimens. The authentic carotenoids used were as follows: Astaxanthin diester, astaxanthin monoester and astaxanthin were extracted and purified from the Antarctic krill Euphausia superba (Yamaguchi et al., 1983; Maoka et al., 1985). Zeaxanthin, diatoxanthin and cynthiaxanthin were extracted and isolated from Spirulina maxima (Miki et al., 1986). RESULTS Typical chromatograms of muscle carotenoids are shown in Fig. 1. (3S,3'S)-Astaxanthin was a dominant carotenoid at the feeding migration stage, while the percentages of zeaxanthin and 4-keto-zeaxanthin increased during spawning migration. However, the amounts of all carotenoids including astaxanthin, zeaxanthin and 4-keto-zeaxanthin decreased during spawning migration (Table 2). The similar behavior of carotenoid composition was observed in both male and female. The serum carotenoid levels markedly increased during spawning migration. Typical chromatograms of serum carotenoids are shown in Fig. 2. (3S,YS)-Astaxanthin was a dominant carotenoid at the spawning migration stage, while zeaxanthin and 4-keto-zeaxanthin as well as astaxanthin markedly

412

SEIICHIANDO and MUTSUO HATANO Table 1. Characteristics of chum salmon specimens Fork

Body

length

weight

Date and locality Stage

Sex

Age of collection

(cm)

(g)

Gonadosomatic

Hepatosomatic

index

index

Male

03

June 27, Ig85. Shizunai coast of

68

4070

0.20

1.99

Female

03

Hokkaido

62

3170

4.59

2.49

Male

03

Sept. 22. 1985. Shibetsu coast of

67

3410

5.88

1.42

Female

03

Hokkaido

69

3800

13.06

2.73

Male

03

Oct. 6, 1985. Lower reaches

68

3010

3.54

2.59

Female

03

(2.4km) of Shibetsu River, Hokkaido

66

3060

20.27

1.43

Male

03

Nov. 19, 1985. Lower reaches

82

5000

1.98

1.38

04

(l.8km) of Yurappu River. Hokkaido

76

3840

1.25

1.67

Feeding migration

Spawning migration

Upstream migration

Spent Female

*(Gonad weight/Body weight) x 100. **(Liver weight/Body weight) x 100.

increased at the upstream migration stage and after spawning. The increase of zeaxanthin and 4-keto-zeaxanthin was particularly found in male serum (Table 3). The carotenoid levels of the ovaries markedly increased during spawning migration. A typical chromatogram of ovaries carotenoids is shown in Fig. 3. (3S,3'S)-Astaxanthin was a dominant carotenoid during spawning migration. The similar behavior of carotenoid composition was observed during spawning migration (Table 4).

in the muscle carotenoids increased and those of astaxanthin decreased during spawning migration. This suggested that astaxanthin was reductively converted to zeaxanthin via 4-keto-zeaxanthin in the muscle. The similar metabolism of muscle carotenoids was observed in both male and female. (2) The reductive metabolic pathways of astaxanthin in the muscle were also confirmed by the results of the carotenoid composition in the serum (Fig. 2 and Table 3). Appreciable amounts of zeaxanthin and 4-keto-zeaxanthin as well as astaxanthin were detected in male serum during spawning migration. The carotenoid metabolism in the DISCUSSION muscle was considered to be responsible for the Several metabolic pathways, based on the contents increase of carotenoids in the serum. The reductive and individual composition of carotenoids and on metabolites of astaxanthin in female serum were their stereochemical grounds, have been proposed. much less than those in male serum, although asThe reductive metabolic pathways of astaxanthin taxanthin was reductively metabolized in the muscle have been suggested in the integuments of yellowtail of both male and female. This indicated that Seriola quinqueradiata (Fujita et al., 1983a; Miki et zeaxanthin and 4-keto-zeaxanthin as well as asaL, 1985) and red sea bream Pagrus major (Fujita et taxanthin in female serum were transported rapidly al., 1983b). Matsuno et al. (1985) have reported the into the integument, since astaxanthin was a domireductive metabolic pathways of (3S,YS)-astaxanthin nant carotenoid in the ovaries during spawning in the eggs of marine fish from the stereochemical migration (Fig. 3 and Table 4). The sexual differences point of view. Kitahara (1983) has suggested the in the transport rate of carotenoids from the muscle following reductive metabolism of astaxanthin in the to the integument via the serum might influence the integument of chum salmon, astaxanthin~4-keto- carotenoid composition in the serum. zeaxanthin~fl-carotene-triol-~zeaxanthin. Thus, the A presumed metabolic pathway, based on the reductive metabolic pathways of carotenoids are contents and individual composition of carotenoids limited to the integuments and eggs of fish. in the muscle, serum and ovaries of chum salmon In this study, however, the reductive metabolic during spawning migration, is given in Fig. 4. This pathways of astaxanthin in the muscle of chum pathway represents a new presumption, the reductive salmon were presumed from the following reasons: metabolism of astaxanthin in the muscle. This indi(1) The muscle carotenoid levels greatly decreased cates that the reductive enzyme systems of carduring spawning migration. If the carotenoids are not otenoids are present not only in the integument but metabolized in the muscle, the similar behavior of also in the muscle. Carotenoid analysis in the incarotenoid composition is observed during spawning tegument is further needed to establish the detailed migration. As shown in Fig. 1 and Table 2, however, carotenoid metabolism in chum salmon during the percentages of zeaxanthin and 4-keto-zeaxanthin spawning migration.

Carotenoid metabolism in chum salmon

413

A

2

I

20

!

I

20

40

I

!

I

40

60

80

I

I

60

80

I

lO0 min

|

lO0 min

Fig. I. HPLC separation of carotenoids extracted from the male chum salmon muscles at the feeding migration stage (A) and after spawning (B). Column, Sumipax OA-2000; Mobile phase, n-hexaneCHjC12-EtOH (50:20:0.5); Flow-rate, 0.8ml/min; Detection, 470nm; Peaks, l = c a n t h a x a n t h i n ; 2 = zeaxanthin; 3 = 4-keto-zeaxanthin; 4 --- diatoxanthin + cynthiaxanthin; 5 = (3R,3'R)-astaxanthin; 6 = ( 3 R , 3 ' S ; m e s o ) - a s t a x a n t h i n ; 7 = (3S,YS)-astaxanthin. Table 2. Content and percentage composition of carotenoids in the chum salmon muscle during spawning migration Feeding migration stage MaI e Total carotenoids (mg / whole muscle)

Female

16.18 (I00) b 12.46 (lO0)

Spawningmigration stage Male

Female

12.33 (I00)

11.24 (100)

Upstreammigration stage Male

FemaI e

0.57 (lO0) 0.48 (100)

0.12 (0.73)

0.04 (0.32)

0.04 (0.32)

0.03 (0.27)

0.19 (I.15)

0.I0 (0.77)

0.32 (2.61)

0.31 (2.80)

0.07 (11.85) 0.04 (8.44)

0.26 (23.75) 0.29 (20.04)

4-Keto-zeaxanthin(3)

0.34 (2.08)

0.20 (1.61)

0.45 (3.69)

0.48 (4.30)

0.03 (4.99) 0.02 (4.27)

0.05 (4.99) 0.05 (3.42)

Diatoxanthin(4) + Cynthiaxanthin(4)

0.06 (0.35)

0.05 (0.37)

0.07 (0.55)

0.12 (1.06)

0.01 (1.53)

0.01 (0.85)

14.52 (89.75) II.42 (91.70)

I0.68 (86.71)i 9.44 (84.00)

1.28 (7.90)

1.65(13.23)

1.28(I0.42) l.O0 (8.90)

(3R,3'S;meso)-Astaxanthin(6)O.07 (0.43)

0.08 (0.67)

0.06 (0.52)

(3S.3'S)-Astaxanthin(7) Unidentified

13.17(81.42) 9.69 (77.80) 0.95 (5.94)

0.65 (5.23)

0.07 (0.84)

tr

tr

(0.54)

1.05 (lO0) 1.44 (lO0)

Zeaxanthin (2)

(3R,3'R)-Astaxanthin(5)

(0.35)

Female

Canthaxanthin(1) a

Astaxanthin fraction

tr

Spent Male

(0.43)

tr

(0.47)

tr

tr

(0.24)

(0.36)

0.42 (73.43) 0.37 (78.56) 0.70 (64.63)1.02 (70.69) 0.04 (6.63) 0.04 (8.94) tr

(tr)

tr

(tr)

O.ll (I0.46) 0.19(12.96) 0.01 (0.58) 0.01 (0.52)

9.34 (75.77) 8.37 (74.46)

0.38 (66.80) 0.33 (69.62) 0.58 (53.59) 0.82 (57.21)

0.77 (6.12)

0.04 (7.85) 0.05 (7.76)

0.86 (7.57)

aPeak numbers on chromatogram. bValues in parentheses represent percentages, with total carotenoids being 100%.

0.03 (5.31) 0.08 (5.25)

414

SEIICHI ANDO and Mu'rsuo HATANO

5

B i

\

A I

I 20

I

I

I

40

60

80

I 1O0 mi n

Fig. 2. HPLC separation of carotenoids extracted from the male chum salmon sera at the spawning migration stage (A) and after spawning (B). HPLC conditions and peak numbers are referred to the legend in Fig. !.

Table 3. Content and percentage composition of carotenoids in the chum salmon serum during spawning migration Spawning migration s t a g e Male Total carotenoids (ng / ml serum)

Female

3298 (lO0) b 4298 (lO0)

Upstreammigration stage Male 9449 (lO0)

Female 8259 (lO0)

Spent Male 6250 (lO0)

Female 8139 (lO0)

Zeaxanthin(2) a

18 (0.55)

47 (l.lO)

816 (8.64)

370 (4.48)

1241 (19.86)

159 (l.gS)

4-Keto-zeaxanthin(3)

85 (2.57)

49 (l.15)

734 (7.77)

446 (5.40)

506 (8.10)

418 (5.13)

Diatoxanthin(4) + Cynthiaxanthin (4)

36 (l.lO)

9 (0.22)

I06 (l.12)

12 (0.14)

61 (0.97)

38 (0.47)

6954 (73.59) 6762 (81.87) 3800 (60.80)

7173 (88.13)

Astaxanthin fraction (3R,3'R)-Astaxanthin(5)

3000 (90.92) 3856(89.68) 469(14.22)

346 (8.04)

879 (9.30)

1514(18.33)

459 (7.34)

(3R,3'S;meso)-Astaxanthin(6) 29 (0.88)

14 (0.33)

52 (0.55)

38 (0.46)

29 (0.46)

(3S,3'S)-Astaxanthin (7) Unidentified

2502 (75.82) 3496(81.31) 159 (4.86)

337 (7.85)

6023 (63.74) 5210(63.08) 3312(53.00) 839 (8.88)

669 (8.11)

*Peak numbers on chromatogram. bValues in parentheses represent percentages, with total carotenoids being 100%.

642 (I0.27)

1403(17.24) 88 (I.08) 5682 (69.81) 351 (4.32)

Carotenoid metabolism in c h u m salmon

I 20

I 40

415

I 60

I 80

I i00 min

Fig. 3. H P L C separation of carotenoids extracted from the chum salmon ovaries at the upstream migration stage. H P L C conditions and peak numbers are referred to the legend in Fig. I.

Table 4. Content and percentage composition of carotenoids in the chum salmon ovaries during spawning migration Feeding migration

Spawningmigration

Upstreammigration

stage

stage

stage

Total carotenoids (mg / whole ovary)

2.510 (100)b

2.580 (I00)

4.863 (lO0)

Canthaxanthin(1) a

0.043 (1.73)

0.016 (0.61)

0.020 (0.41)

Zeaxanthin(2)

0.056 (2.24)

0.137 (5.31)

0.070 (1.44)

4-Keto-zeaxanthin(3)

0.049 (I.94)

0.164 (6.34)

0.193 (3.97)

Diatoxanthin(4) + Cynthiaxanthin(4)

0.011 (0.43)

0.034 (1.30)

0.046 (0.95)

2.181 (86.90)

2.109 (81.75)

4.276 (87.92)

0.292(11.64)

0.251 (9.73)

0.473 (9.72)

(3R,3'S;meso)-Astaxanthin(6) 0.014 (0.54)

0.014 (0.53)

0.032 (0.66)

(3S,3'S)-Astaxanthin(7)

1.875(74.72)

1.844(71.49)

3.771(77.54)

0.170 (6.76)

0.121 (4.69)

0.258 (5.31)

Astaxanthin fraction (3R,3'R)-Astaxanthin(5)

Unidentified

"Peak numbers on chromatogram. bValues in parentheses represent percentages, with total carotenoids being 100%. C.B.P. S7/2B~N

416

"SEncHI ANDO and Mtrrsuo HATANO

Muscle Astaxanthin

Serum

--

) Astaxanthin

I

4-Keto-zeaxanthinn mmm~4-Keto-zeaxanthin

Zeaxa~nthin

Zeaxanthin

Ovary Astaxanthin

)4-Keto-zeaxanthin

Zeaxanthin

1 Reductive reaction Oxidative reaction Esterification reaction Integument Fig. 4. A presumed metabolic pathway of carotenoids in the chum salmon during spawning migration. ml~, main pathway; ~ , supplemental pathway.

Acknowledgements--The authors wish to thank Professor S. Konosu for facilities of HPLC. The authors are indebted to Dr K. Yamaguchi for his valuable discussion and to Mr T. Mori for his excellent technical assistance. REFERENCES Ando S. and Hatano M. (1986c) Carotenoids in an egg yolk protein of chum salmon ( Oncorhynchus keta ). Agric. biol. Chem. 50, 1043-1044. Ando S. and Hatano M. (1986d) Deterioration of chum salmon Oneorhynehus keta muscle during spawning migration--XIV. Carotenoids in the serum lipoproteins of chum salmon associated with migration. Bull. Fac. Fish. Hokkaido Univ. 37, 148 156. Ando S., Takeyama T., Hatano M. and Zama K. (1985) Carotenoid-carrying lipoproteins in the serum of chum salmon (Oncorhynchus keta) associated with migration. Agric. biol. Chem. 49, 2185-2187. Ando S., Takeyama T. and Hatano M. (1986a) Transport associated with serum vitellogenin of carotenoid in chum salmon (Oncorhynchus keta). Agric. biol. Chem. 50, 557-563. Ando S., Takeyama T. and Hatano M. (1986b) Isolation and characterization of a carotenoid-carrying lipoprotein in the serum of chum salmon (Oncorhynchus keta) during spawning migration. Agric. biol. Chem. 50, 907-914. 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. Fujita T., Satake M., Hikichi S., Takeda M., Shimeno S., Kuwabara H., Miki W., Yamaguchi K. and Konosu S. (1983a) Pigmentation of cultured yellowtail with krill oil. Bull. Jap. Soc. scient. Fish. 49, 1595-1600. Fujita T., Satake M., Watanabe T., Kitajima C., Miki W., Yamaguchi K. and Konosu S. (1983b) Pigmentation of cultured red sea bream with astaxanthin diester purified from krill oil. Bull. Jap. Soc. scient. Fish. 49, 1855-1861. Kitahara T. (1983) Behavior of carotenoids in the chum salmon (Oncorhynchus keta) during anadromous migration. Comp. Biochem. Physiol. 76B, 97-101.

Maoka T., Katsuyama M., Kaneko N. and Matsuno T. (1985) Stereochemical investigation of carotenoids in the Antarctic krill Euphausia superba. Bull. Jap. Soc. scient. Fish. 51, 1671-1673. Matsuno T., Katsuyama M. and Nagata S. (1980) Comparative biochemical studies of carotenoids in fishes-XIX. Carotenoids of chum salmon, Biwa trout, red-spotted masu salmon, masu salmon, and kokanee. Bull Jap. Soc. scient. Fish. 46, 879-884. Matsuno T., Maoka T., Katsuyama M., Ookubo M., Katagiri K. and Jimura H. (1984) The occurrence of enantiometric and meso-astaxanthin in aquatic animals. Bull. Jap. Soc. scient. Fish. 50, 1589-1592. Matsuno T., Katsuyama M., Maoka T., Hirono T. and Komori T. (1985) Reductive metabolic pathways of carotenoids in fish (3S,YS)-astaxanthin to tunaxanthin A, B and C. Comp. Biochem. Physiol. 80B, 779-789. Miki W., Yamaguchi K., Konosu S., Takane T., Satake M., Fujita T., Kuwabara H., Shimeno S. and Takeda M. (1985) Origin of tunaxanthin in the integument of yellowtail (Seriola quinqueradiata). Comp. Biochem. Physiol. 80B, 195-201. Miki W., Yamaguchi K. and Konosu S. (1986) Carotenoid composition of Spirulina maxima. Bull. Jap. Soc. scient. Fish. 52, 1225-1227. Nakamura K., Hata M. and Hata M. (1985) A study on astaxanthin in salmon Oncorhynchus keta serum. Bull. Jap. Soc. scient. Fish. 51, 979-983. Schiedt K., Leuenberger F. J. and Vecchi M. (1981) Natural occurrence of enantiometric and meso-astaxanthin. 5. Ex wild salmon (Salmo salar and Oncorhynehus ). Helv. Chim. Aeta 64, 449-457. Torrissen O. J. (1985) Pigmentation of saimonids: Factors affecting carotenoid deposition in rainbow trout (Salmo gairdneri). Aquaculture 46, 133-142. Torrissen O. J. and Naevdal G. (1984) Pigmentation of salmonids-genetical variation in carotenoid deposition in rainbow trout. Aquaculture 38, 59-66. Yamaguchi K., Miki W., Toriu N., Kondo Y., Murakami M., Konosu S., Satake M. and Fujita T. (1983) The composition of carotenoid pigments in the Antarctic krill Euphausia superba. Bull. Jap. Soc. scient. Fish. 49, 1411-1415.