106B, No. 2, pp. 243-250, 1993. 0305-0491/93 $6.00 + 0.00. Printed in Great Britain ... M. modiolus is characterized by a high proportion (86-90% of total carotenoids) of aeetylenic ..... 15.6 Hz),6.38(dd, H-14,J = 11.0 Hz,J = 14.2 Hz),. 6.47 (d ...
Comp. Biochem. PhysioLVol. 106B,No. 2, pp. 243-250, 1993 Printed in Great Britain
0305-0491/93 $6.00+ 0.00 © 1993PergamonPress Ltd
CAROTENOIDS IN FOOD CHAIN STUDIES---V. CAROTENOIDS OF THE BIVALVES MODIOLUS MODIOLUS A N D PECTEN MAXIMUS--STRUCTURAL, METABOLIC AND FOOD CHAIN ASPECTS B. BJERKENG*, S. HERTZBERGt and S. LIAAEN-JENSEN Organic Chemistry Laboratories, Norwegian Institute of Technology, University of Trondheim-NTH, N-7034 Trondheim-NTH, Norway
(Received 5 March 1993; accepted 7 April 1993) Abstract--l. The quantitative and qualitative carotenoid composition of two harvests of the mussel Modiolus modiolus, is reported. Sixteen different carotenoids were identified, employing TLC, HPLC, VISand mass spectra; occasionally IR, ~H NMR and CD, and derivatization to carbamates or camphanates for assignment of chirality. 2. M. modiolus is characterized by a high proportion (86-90% of total carotenoids) of aeetylenic carotenoids. 3. The carotenoid composition is compatible with microalgal diets of diatoms and dinoflagellates. 4. The metabolism of allenic carotenoids (fucoxanthin and peridinin) to acetylenic carotenols resembled that of Mytilus edulis. 5. A 4-keto oxidative pathway of diatoxanthin to pectenolone and of (3R,YR)-zeaxanthin to (3S, YS)-astaxanthin is deduced. 6. Quantitative analysis revealed the presence of C37-skeletal allenic carotenoids (50% of total) indicating mainly a dinoflagellate diet in the scallop Pecten maximus. Individual 4(4")-keto-C40-carotenoids indicated the operation of an oxidative metabolic pathway.
carotenoid composition of M. modiolus is not previously reported. Also included is a quantitative examination of the carotenoids of Pecten maximus, a scallop also feeding on phytoplankton.
INTRODUCTION
Shellfish is popular seafood with high potentials for Norwegian aquaculture. The scallop P. maximus is a promising species for shellfish farming, while the mussel M. modiolus probably is best suited for harvesting at sea due to its low growth rate. Recently we have examined the carotenoid composition of Mytilus edulis (edible mussel) in great detail (Hertzberg et al., 1988) and carried out feeding experiments with unialgal cultures of established carotenoid composition (Partali et al., 1989). As a result, knowledge has been gained on the resorption and metabolic conversion of microalgal carotenoids by M. edulis. In particular (a) hydrolysis of acetates, (b) conversion of allenic to acetylenic end groups and (¢) reductive opening of carotenoid 5,6-epoxides to 5,6-glycols were found to be general metabolic processes (Partali et al., 1989). M. modiolus is known to be a filter feeder with similar microalgal diet to Mytilus edulis. This investigation on the carotenoid composition of M. modiolus was carried out with food chain aspects in mind. The
MATERIALS AND METHODS
Biological materials Modiolus modiolus harvested from the Trondheimsfjord was used. Harvest 1 (March 1985) consisted of three specimen of M. modiolus (acetoneextracted dry weight 40 g) and provided in total 11 mg carotenoids (0.03%). Harvest 2 (August 1989) contained seven mussels of M. modiolus purchased at the local fish market (acetone-extracted dry weight 142 g) and provided 68 mg carotenoids (0.05%). Six specimen of Pecten maximus (total wet weight 261 g after removal of mantles) was purchased at the local fish market, August 1989. The yield was less than 5 mg crude carotenoids (ca 0.02% based on the acetone-extracted dry weight) prior to chromatographic purification. Carotenoid isolation procedure
*Present address: NORCONSERV, Institute of Fish Processing and Preservation Technology, P.O. Box 327, N-4001 Stavanger, Norway. tPresent address: Laboratory of Biotechnology, Norwegian Institute of Technology, University of Trondheim-NTH, N-7034 Trondheim, Norway.
General precautions for work with carotenoids were taken. Instruments and methods used were as commonly employed in our laboratory (LiaaenJensen and Jensen, 1965; Grung et al., 1989). The mussels were cleaned, and colourless parts removed.
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B. BJERKENGet al.
The fresh mussels or scallops were minced and extracted with acetone/methanol (7:3), followed by extraction with acetone in a Waring blender at room temperature until the residue was colourless. The extracts contained considerable amounts of decomposed chlorophylls. No saponification step was included. Column chromatography was carried out on silica with increasing amounts of ether in hexane as mobile phase, TLC on silica plates or on alkaline plates (Bj~rnland, 1985) with trichloroethane/MeOH (100: 5, System 1), trichloroethane/acetone/isopropanol (100:20:5, System II) and various other eluents. When not otherwise stated, silica plates were used. HPLC was carried out isocratically on a Spherisorb S-5CN nitrile (250 x 4.6 mm; 5 pm particles; System III) or a Spherisorb 5 Si Techsphere silica column (250 x 4.6 mm; 5 #m particles; System IV) with hexane/isopropyl acetate/acetone/methanol (77 : 17:7: 0. I). Spectral fine-structures for VIS spectra are expressed as %Ill/11 (Ke et al., 1970). For mass spectra only prominent or diagnostically useful ions are cited. CD spectra were recorded in EPA (diethyl ether/2-methylbutane/ethanol 5: 5 : 2).
Characterization of individual carotenoids The quantitative carotenoid composition, established by TLC, co-chromatography tests, VIS-spectra and MS, is given in Table 2, listing the carotenoids in order of increasing adsorption.
Individual carotenoids of M. modiolus fl,fl-Carotene (1). Available 0.26 mg. Rf = 0.93, silica (20% acetone in heptane); VIS 2max n m (hexane); (420), 448 and 474, %1II/11 = 24; MS (210°C) m/z (%) 536 ([M] +, 100). Eehinenone (2). Available 0.14 rag. Rr = 0.38 silica (20% acetone in heptane), Rf = 0.63 alkaline plates (20% acetone in heptane); VIS 2maxnm (acetone) 457, (473); MS (210°C) m/z (%) 550 ([M] +, 100). Unidentified 1. R f = 0.47 (System I), unchanged after saponification; VIS 2~axnm (hexane) 468, (acetone) 470; MS (200°C) unsuccessful. Anhydro-zeaxanthin (3)-like. Available 0.03mg. Rf=0.28 (System I); VIS 2maxnm (hexane) 450, (acetone) 456; MS (210°C) m/z (%) 550 ([M] ÷, 32), 273 (64), 271 (100). HPLC (nitrile or silica columns) revealed the presence of two stereoisomers. Isomer 1 VIS 2m~xnm (430) 458 and 483 (61% of total), RT = 9.43 min and Isomer 2 VIS )'maxnm (in eluent) 458 (36%), RT = 10.01 min upon co-chromatography with echinenone (2) RT = 5.2 min. The eluent was hexane/isopropyl acetate/ethyleneglycol monomethyl ether: N-ethyl diisopropylamine 88 : 12: 0.2: 0.1. 3',4'-Anhydro-pectenolone (4)-like. Available 0.09 mg. R f = 0.22, silica (20% acetone in heptane), Rf = 0.46, alkaline plates (30% acetone in heptane); VIS 2m~Xnm (acetone) 464, (480); MS (210°C) m/z (%) 562 ([M] ÷, 100); 560 ([M-2] ÷ , 20); 546 ([M-16] ÷ , 17); 544 ([M-18] +, 6); 470 ([M-92] ÷, 5); 281 ([M] :+, 4); FT-IR (KBr) cm -l 3395 m (OH), 2170 w (C = C),
1662 m (conj. C = O); CD (EPA) AE 224 (2.9), 240 (0), 285 (--2.9), 300 (0), 324 (1.5), 345 (0), 375 (--1.4), 401 (0). Pyrrhoxanthin (5). Available 0.05mg. R f = 0.11 silica (20% acetone in heptane), Rf= 0.41 alkaline plates (30% acetone in heptane); VIS 2maxnm (acetone) (473) 485; MS (210°C) m/z (%) 612 ([M] +, 18) 594 ([M- 18] +, 14), 576 ([M- 18-18] +, 8), 197 (100). Unidentified 2. Available 0.09 mg. Rf = 0.47, silica (40% acetone in heptane); HPLC R v = 7.74 (System IV, flow 2.0ml/min); VIS 2max nm (acetone) 485, (512); MS (210°C) unsuccessful. (3R, 3'R)-Zeaxanthin (6). Available 0.25. Rf = 0.30 silica (40% acetone in heptane). VIS 2maxnm (acetone) (425) 450 and 478; MS (210°C) m/z (%) 568 ([M] +, 100), 550 ([M-18] +, 7), 476 ([M-92] ÷, 14). 6 was converted to the dicarbamate (Partali et al., 1989) and HPLC (silica, hexane/isopropyl acetate/ ethyleneglycol monomethyl ether/N-ethyl diisopropylamine 88:12:0.2:0.1) in direct comparison with authentic 6-dicarbamate, confirmed the presence of the optically pure (3R, 3'R) isomer. Diatoxanthin (7). Available 0.63 mg. TLC Rf= 0.28 silica (40% acetone in heptane), Rf=0.31 alkaline plates (30% acetone in heptane); HPLC RT = 10.83 (System IV, flow 2.0 ml/min). VIS J'maxnm (acetone) (425) 452 and 480; MS (210°C) m/z (%) 566 ([M] +, 100), 551 ([M-15] +, 6), 548 ([M-18] +, 6), 474 ([M-92] ÷, 2), 283 ([M] 2+, 17), 43 (100). 500MHz 1HNMR (CDC13), (6-values relative to CHC13 = 7.26ppm) 1.07 (s, Me-16' and Me-IT), 1.15 (s, Me-16), 1.20 (s, Me-17), 1.46 (dd, H-2 ax), 1.48 (dd, H-Tax), 1.74 (s, Me-18'), 1.77 (m, H-2' eq), 1.83 (dd, H-2 eq), 1.92 (s, Me-18), 1.97 (s, Me-19',20'), 1.98 (s, Me-20), 2.00 (s, Me-19), 2.05 (dd, H-4' ax), 2.06 (dd, H-4 ax), 2.39 (dd, H-4' eq), 2.43 (dd, H-4 eq), 6.10 (d, H-7', J = 14.6Hz), 6.13 (d, H-10'), 6.15 (d, H-8', J = 14.6 Hz), 6.2545.27 (m, H-14, 14'), 6.35 (d, H-IT, J = 14.2 Hz), 6.36 (d, H-12, J = 14.3 Hz), 6.51 (dd, H-11, J = 11.5 Hz, J = 14.3 Hz), ca 6.00--6.67 (H-15, H-15', H-IV); FT-IR (0.2 mg KBr) cm -~ ca 3400 (m, OH), 2169 w (C = C). (3S, 3'S)-Astaxanthin (8). Available 0.56 mg. TLC Rf--0.31 silica (40% acetone in heptane), R f = 0.36 alkaline plates (30% acetone in heptane); HPLC R T --- 7.34 (System III, flow 1.5 ml/min). VIS 2m~xnm (acetone) 473; MS (210°C) m/z (%) 596 ([M] ÷, 100), 594 ([M-2] ÷, 15), 580 ([M-16] ÷, 11), 578 ([M-18] +, 15). 500 MHz ~HNMR (CDCI3) , (6-values relative to CHC13 = 7.26 ppm) 1.21 (s, Me-16 and 16'), 1.32 (s, Me-17, 17'), 1.81 (t, H-2 and 2' ax), 1.94 (s, Me-18, 18'), 1.99 (s, Me-19 and 19'), 2.00 (s, Me-20 and 20'), 2.16 (dd, H-2 and 2' eq, Jvic= 5.7 Hz, Jser~= 11.4 Hz), 4.32 (m, H-3 and H3' ax), 6.22 (d, H-7 and H-7'), 6.30 (d, H-10 and H-10' and H-14 and H-14'), 6.43 (d, H-8 and H-8', J = 16.1 Hz), 6.45 (d, H-12 and H-IT, J = 15.4 Hz), 6.66 (dd; H-11 and H-11', J --- 11.4 Hz, J = 15.6Hz), 6.67 (m, H-15 and 15'). F T - I R ( 0 . 2 m g K B r ) c m -1 3424w (OH), 1654s (conj. C = O). Assignments were made according to re-
Carotenoids in food chains of bivalve molluscs ported data (Englert, 1982). Co-chromatography with authentic 8_ on TLC and HPLC gave no separation. Acetylation provided the diacetate, MS (210°C) m/z 680 (M + ). Dicamphanates of_8 coeluted with the (3S, YS)-isomer (92% all-trans- and 8% c/s) of the dicamphanates prepared from astaxanthin in "Carophyll pink" (1 : 2:1-mixture of the (3R,3'R)-, (3R,YS)- and (3S, YS)-_8 isomers, respectively) Hoffmann-La Roche, Basel, Switzerland. 7,8-Didehydro-astaxanthin (9). Inseparable from 8 on silica plates, but more strongly adsorbed on alkaline plates (System 1, ca 10% of 8); VIS 2m~xnm (acetone) 473; MS (210°C) in mixture with _8m/z (%) 594 ([M] + ) (32% of m/z 596). (3S,3"R)-Pectenolone (10). Available 5.35 mg. Rr = 0.28 silica (40% acetone in heptane), Rf = 0.34 alkaline plates (30% acetone in heptane); HPLC Rx = 7.36 min. (System III, flow 1.5 ml/min), R x = 12.12rain. (System IV, flow 2.0ml/min); VIS 2 ~ nm (acetone) 458 (475); MS (210°C) m/z (%) 580 ([M] +, 100), 578 ([M-2] +, 28), 564 ([M-16] + , 6), 562 ([M-18] +, 5), 488 ([M-92] +, 3), 474 ([M-106] +, 2), 426 ([M-154] +, 4), 290 ([M] 2+, 2); 500 MHz ' H N M R (CDC13), (6-values relative to CHCI3= 7.26ppm) 1.15 (s, Me-16'), 1.21 (s, Me-IT), 1.21 (s, Me-16), 1.33 (s, Me-17), 1.46 (m, H-2' ax), 1.81 (m, H-2 ax), 1.84 (d, H-2' eq), 1.92 (s, Me-18'), 1.94 (s, Me-18), 1.96 (s, Me-20'), 1.98 (s, Me-20, and 19'), 2.00 (s, Me-19), 2.06 (dd, H-4' ax), 2.15 (dd, H-2 eq), 2.42 (dd, H4'eq), 3.98 (m, H-Y), 4.32 (m, a-3), 6.21 (d, a-7; J = 16.0 Hz), 6.27-6.67 (m, olefinic protons); FT-IR (0.2mgKBr) cm -1 2169w ( C = C ) , 1662s (conj. C = O); CD (EPA) AE 229 (0); 239 (--2.1); 252 (0); 267 (2.1); 274 (0); 307 (--5.1); 396 (0). 9,9'-Di-cis-alloxanthin (lib). Available 0.38mg. TLC Rf=0.43 silica (50% acetone in heptane), R f = 0.78 alkaline plates (50% acetone in heptane). HPLC R x = 6.93 min. (System III, flow 1.5 ml/min); VIS 2maxnm (acetone) (419), 453 and 481, %11I/I1 = 42; MS (210°C) m/z (%) 564 ([M] +, 100), 562 ([M-2] +, 4), 549 ([M-15] +, 3), 548 ([M-16] +, 2), 546 ([M-18] +, 2), 472 ([M-92] +, 1), 457 ([M-I 5-92] + , 1), 410 ([M-154] +, 1), 282 ([M] 2+, 44), 43 (47); 500MHz I H N M R (CDC13) , (t~-values relative CHCI3 = 7.26ppm ) 1.19 (s, Me-16 and 16'), 1.27 (s, Me-17 and 17'), 1.48 (dd, H-2 and 2' ax), 1.85 (dd, H-2 and 2' eq), 1.93 (s, Me-18 and 18'), 1.97 (s, Me-20 and 20'), 2.00 (s, Me-19 and 19'), 2.09 (dd, H-4 and 4' ax), 2.45 (m, H-4 and 4' eq); 4.01 (m, H-3 and Y), 6.25 (d, H-14 and 14'; J = 8 Hz), 6.29 (d, H-10 and 10'; J = 11.0 Hz), 6.35 (d, H-12 and 12'; J = 15.3 Hz), 6.61 (m, H-15 and 15'); 6.83 (dd, H-11 and 11'; J = 11.2 Hz, J = 15.2 Hz), FT-IR (KBr) cm -1 3376 w (OH), 2177 w (C - C). (3R,YR)-Alloxanthin (lla). Available 3.88mg. TLC Rf=0.41 silica (40% acetone in heptane), R f = 0.73 alkaline plates (50% acetone in heptane); HPLC Rx = 7.18 min. (System III, flow 1.5 ml/min), R T = 11.99 rain. (System IV, flow 2.0ml/min); VIS 2 ~ nm (acetone) (420), 452 and 482, %11I/II = 42; CBPB 106/2--B
245
MS (210°C) m/z (%) 564 ([M] +, 100), 562 ([M-2] +, 9), 560 ([M-4] +, 1), 549 ([M-15] +, 2), 548 ([M-16] +, 1), 546 ([M-181 +, 1), 536 ([M-28] +, 1), 532 ([M-1517] +, 1), 472 ([M-92] +, 1), 457 ([M-15-92] +, 1), 439 ([M-15-18-92] +, 0.6), 410 ([M-154] +, 2), 282 ([M] 2+, 26); 43 (22); 500 MHz IHMNR (CDCI3), (3-values relative CHC13=7.26ppm) 1.14 (s, H-16 and 16'), 1.20 (s H-17 and 17'), ca 1.43 (broad s, 3- and 3' OH), 1.46 (m, H-2 and 2' ax), 1.83 (m, H-2 and 2' eq), 1.92 (s, Me-18 and 18'), 1.96 (s, Me-20 and 20'), 2.00 (s, Me-19 and 19'), 2.06 (dd, H-4 and 4' ax), 3.99 (m, H-3 and Y), 6.27 (d, H-14 and 14'), 6.36 (d, H-12 and 12'; J = 14.4Hz), 6.45 (d, H-10 and 10'), 6.52 (dd, H-11 and 11'; J = 14.5 Hz, J = 11.5 Hz), 6.64 (m, H-15 and 15'). Assignments were made according to published values for the end group including H-11 (Englert, 1985); F T - I R (0.2 mg KBr) cm -l 3 3 9 0 m (OH), 2171w (C--C); CD (EPA) AE 244 (--2.6), 262 ( - l . 1 ) , 290 (-3.0), 362 (0). Mytiloxanthin (12). TLC Rf=0.36 (System 1), inseparable from 1__2ex Mytilus edulis; VIS 2mxnm (acetone) 468 and (495), (acetone) 470; MS (210°C) m/z (%) 598 ([M] +, 100), 580 ([M-18] +, 9), 506 ([M-92] +, ll), 197 (60), 179 (56), 149 (83). Acetylation provided the presumed diacetate Rf=0.91 (System l, 9'-cis?) and Rf = 0.86 (System l, all-trans ?). Peridinin (13). Available 0.14mg. TLC Rf=0.36 silica (40% acetone in heptane), Rf= 0.69 alkaline plates (50% acetone in heptane). HPLC: 15.90 min. (System III, flow 1.5 ml/min); VIS 2m~ nm (acetone) 455 and (475); MS (230°C) m/z (%) 630 ([M] +, 56), 614 ([M-16] +, 9), 612 ([M-18] +, 25), 596 ([M16-18] +, 5), 570 ([M-60] +, 6), 558 ([M-18-44] +, 6), 552 ([M-18-60] +, 12), 212 (47), 197 (100), 181 (83). Pyrrhoxanthinol (14). Available 0.40mg. TLC Rr = 0.44 silica (50% acetone in heptane), R f = 0.68 alkaline plates (50% acetone in heptane); VIS 2max nm (acetone) 454 and (473); MS (230°C) m/z (%) 570 ([M] +, 47), 554 ([M-16] +, 7), 552 ([M-18] +, 6), 508 ([M-18-44] +, 5), 478 (9), 181 (100). Pectenol (15). Available 0.12 mg. TLC Rf = 0.22 silica (50% acetone in heptane), R f = 0.54 alkaline plates (50% acetone in heptane); VIS 2m~nm (acetone) (425), 447 and 475, %III/II = 38; MS (210°C) m/z (%) 582 ([M] +, 100), 564 ([M-18] +, 24), 548 ([M-16-18] +, 11), 546 ([M-18-18] +, 5), 490 ([M92] + , 5). Peridinol (16). Available 0.09 mg. TLC Rf = 0.15 silica (50% acetone in heptane), Rf= 0.50 alkaline plates (50% acetone in heptane); VIS 2m~nm (acetone) 453 and (475); MS (210°C) m/z (%) 588.3461 (C39H4806, calculated:588.3451) ([M]+, 100), 570 ([M-18] +, 27), 552 ([M-18-18] +, 20), 478 ([M-110] +, 39), 221 (46); 181 (89).
Individual earotenoids of P. maximus fl,fl-Carotene (1). Available 0.064mg. Rf=0.93 (20% acetone in heptane), VIS )'maxnm (acetone) 447,
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B. BJE~r.Er~Get al.
472, %III/II = 24. MS (210°C) m/z (%) 536 ([M] +, 100). Echinenone (2). Available 0.039mg. Rf=0.38 (20% acetone in heptane), VIS 2m~xnm (acetone) 452. MS (210°C) rn/z (%) 550 ([M]+, 100). (3S,YS)-Astaxanthin (8). Available 0.024 mg. VIS 2m~ nm (acetone) 473. TLC (silica, 40% acetone in heptane) Rf= 0.31, (alkaline plates, 30% acetone in heptane) Rf=0.36; HPLC: Rr=7.34 (System I, 1.5 ml/min). Co-chromatography with authentic 8 on TLC and HPLC. Dicamphanates of 8 co-chromatographed with the (3S, YS)-isomer of the dicamphanates prepared from "Carophyll pink", Hoffmann-La Roche, Basel, Switzerland. TLC (alkaline plates, 30% acetone in heptane) Rr = 0.35; VIS 2 ~ n m (acetone) 473. MS (210°C) m/z (%) 596 ([M] ÷, 100), 594 ([M-2] ÷ , 43), 580 ([M-16] ÷ , 83), 578 ([M-18] ÷, 38), 504 ([M-92] ÷, 2), 490 ([M-106] +, 1). Pectenolone (10). Available 0.31 mg. VIS 2maxnm (acetone) 462, (480). TLC (silica, 40% acetone in heptane) Rf = 0.28, (alkaline plates, 30% acetone in h e p t a n e ) R f - - 0 . 3 4 . MS (210°C) m/z (%) 580 ([M] +,
594 ([M-18-18] ÷, 2), 586 ([M-44]+, 0.2), 570 ([M-60] ÷, 3), 552 ([M-18-60] +, 15), 550 ([M-80] +, 2), 538 ([M-92]+, 4), 534 ([M-18qS-60] +, 2), 487 ([M-143] +, 3), 358 ([M-272] ÷, 9), 234 (23), 223 (26), 212 (37), 197 (100), 181 (76). 500 MHz tH-NMR (CDCI3), (f-values relative to CHCI3 =7.26), 0.97 (s, Me-16'), 1.07 (s, Me-16), 1.19 (s, Me-IT), 1.20 (s, Me-18'), 1.35 (s, Me-17), 1.38 (s, Me-18), 1.64-1.69 (H-2', H-4'), 1.83 (s, Me-19), 2.07 (s, Me in acetyl), 2.26 (Me-20'), 2.32 (m, H-4), 2.40 (m, H-4'), 3.91 (m, H-Y), 5.38 (m, H-3), 5.73 (s, H-12'), 6.05 (s, H-8), 6.11 (d, H-10, J = l l . 7 H z ) , 6.37 (d, H-8', J = 15.6 Hz),6.38(dd, H-14,J = 11.0 H z , J = 14.2 Hz), 6.47 (d, H-14', J = l l . 7 H z ) , 6.51 (dd, H-12, J = 14.3 Hz, J = ll.0Hz), 6.61 (dd, H-11, H-15'), 7.02 (s, H-10'), 7.17 (d, H-7', J=15.6Hz). Assignments were made in comparison with reported data (Skjetne et al., 1984; Krane et al., 1992). FT-IR (64scan, 0.2mg in KBr)crn -1 3420 (m, OH); 2957, 2924, 2853 (s, CH-), 1928 (w, -C----C-----C-), 1739 (s, -C-----O,acetate and butenolide), 1521 (m, -C-----C-), 1456 (m, CH2), 1378, 1365 (m, gem. 100), 578 ([M-2] +, 17), 564 ([M-16] ÷, 6), 562 (IM- CH3), 1247 (m, C-O-, ester), 1182, 1163, 1124 (m, 18] 4, 4), 488 ([M-92] ÷, 2), 474 ([M-106] ÷, 2), 290 C-O). ([M] 2+, 7). Pectenol (15). Available 0.045 mg. TLC (silica, Alloxanthin (11). Available 0.096 mg. TLC (silica, 40% acetone in heptane) Rf----0.35, (alkaline plates, 40% acetone in heptane) Rf= 0.41, (alkaline plates, 40% acetone in heptane) Rf=0.53. VIS 2 ~ n m 50% acetone in heptane) R f = 0.73. VIS 2m~xnm (acetone) 458, (480). MS (210°C) m/z (%) 582 ([M] +, (acetone) (420), 452, 482; %111/I1= 42. MS (210°C) 100), 564 ([M-18] ÷, 18), 291 ([M]2÷, 10). m/z (%) 564 ([M]÷, 100), 549 ([M-15] ÷, 7), 472 Peridinol (16). Available 0.026 mg. TLC (silica, ([M-92] +, 4), 282 ([M]2÷, 2). 50% acetone in heptane) Rf = 0.15, (alkaline plates, Peridinin (13). Available 0.79 mg. TLC (silica, 40% 50% acetone in heptane) Rf= 0.50. VIS 2m,xnm acetone in heptane) Rf=0.36, (alkaline plates, (acetone 453, (475). MS (210°C) m/z (%) 588 50% acetone in heptane) Rf=0.69; HPLC: ([M] +, 33), 570 ([M-18] +, 31), 552 ([M-18-18] ÷, 8), RT = 15.90 min, (System I, 1.5 ml/min). VIS 2maxnm 528 ([M-60] ÷, 4), 510 ([M-18-60] ÷, 5), 221 [homo(acetone) 462, (475). MS (210°C) m/z (%) 630 pyrrylium] ÷ (36), 197 (72), 181 [pyrrylium] + ([M] ÷, 33), 612 ([M-18] +, 36), 596 ([M-16-18] ÷, 4), (10o).
Table 1. Carotenoids of Modiolus modiolus In % total carotenoids Harvest 1" Presumed microalgal metabolic precursor p,fl-Carotene (1) Diatoxanthin (7)
Fucoxanthin (17) Peridinin (13)
Zeaxanthin (6) Unknown Total carotenoid (%) *March 1985. tAugust 1989.
Carotenoid isolated
Non-aeetylenic
fl,fl-carotene (1) Echinenone (2) Diatoxanthin (7) Pectenol (15) Pectenolone (10) 7,8-Didehydro-astaxanthin (9) Y,4'-Anbydro-pectenolone (4) Mytiloxanthin (12) Alloxanthin (11) Peridinin (13) Peridinol (16) Pyrrboxanthin (5) Pyrrhoxanthinol (14) Zeaxanthin (6) Anhydro-zeaxanthin (3) Astaxanthin (8) Unidentified 1 Unidentified 2
Harvest 2t Acetylenic
Non-acetylenie
28
Aeetylenic
5 1
16 0.5
50 0.6
5 36
31 1
0.1 0.5 3
(0.5) 14
86 (0.5)
8
(0.8) 90 (0.8)
Carotenoids in food chains of bivalve molluscs RESULTS AND DISCUSSION
Carotenoid structures and metabolism
Assuming that the carotenoid composition was dependent on the previous microalgal diet, two harvests of M. modiolus from different seasons were examined. The quantitative carotenoid composition is presented in Table 1, with reference to presumed microalgal carotenoid precursors. The individual carontenoids were identified on the basis of Rf-values and co-chromatography tests with authentic compounds when available, absorption spectra in the visible region (VIS), CD, FT-IR and mass spectra (MS), and when sample size permitting also ~H N M R spectra. Chemical derivatizations, ineluding acetylation and carbamate (Riittimann et al., 1983) and camphanate (Vecchi and M/iller, 1979) formation for diastereomeric resolution, were effected. The fifteen different carotenoids identified in M. modiolus are treated in approximate order of increasing polarity; see Scheme 1 for structures. fl,fl-Carotene (1) was characterized by VIS and MS data, and echinenone (2) by VIS, MS and co-chromatography with an authentic reference. Anhydro-zeaxanthin (3)-like was tentatively identified by VIS, MS and relative RT. A minor carotenoid had MW = 582, compatible with the molecular formula C40H5002. The mass spectrum showed high intensity [M-2] +- and [M-16] +-ions typical for ~t-ketols (Enzell and Wahlberg, 1980). The weak absorption at 2170 cm -t in the FT-IR-spectrum (S~rensen et al., 1968) and the high Cotton effect in the CDspectrum (Sturtzenegger et al., 1980) indicated a long distance between the carbonyl and acetylenic functions. This carotenoid was tentatively assigned the structure of Y,4'-anhydro-pectenolone (4), not previously described. Pyrrhoxanthin (5) was characterized by VIS-data and MS. Optically pure (3R,3'R)-zeaxanthin (6) was identified by VIS- and MS-data and by co-chromatography (HPLC) of the dicarbamate (R/ittimann et al., 1983) with authentic references. Diatoxanthin (7) was identified by VISdata, MS and co-chromatography with an authentic sample. Optically pure (3S, YS)-astaxanthin (8) was identified by VIS-data, MS, MS of the diacetate and HPLC of the dicamphanate (Vecchi and Miiller, 1979) in comparison with authentic reference compounds. 7,8-Didehydroastaxanthin (9), a minor constituent more strongly adsorbed than 8 on alkaline plates, was also detected by MS in mixture with 8. Pectenolone (10) was characterized by Rr-values, VIS-data, MS, and ~H N M R (500 MHz), FT-IR and CD. Chiroptical data were qualitatively consistent with reported spectra (Hiraoka et al., 1982) and compatible with the (3 S, YR)-configuration. (3R,3"R)-Alloxanthin (11) was isolated as the alltrans-(lla) and the thermodynamically more stable 9,9'-dic/s (lib) isomer. Both isomers (lla,b) were individually characterized by VIS, MS, ~H N M R
247
(500 MHz) and CD. The IH NMR-data presented for l l a are consistent with the data presented by Davies et al. (1984), although the 6-values for H-3 and H-Y assigned here is more compatible with the structure of l l a (Englert, 1985). Mytiloxanthin (12), inseparable from 12 (3R,3'S,5'R) (Chopra et al., 1988) ex Mytilus edulis, was characterized by VIS-data, MS and the formation of a diacetate. Peridinin (13), inseparable from authentic 13, was further characterized by VIS data and MS, pyrrhoxanthinol (14) by VIS-data, MS (high resolution) and Rx-value, pectenol (15) by VIS-data and MS and peridinol (16) by VIS-data, MS and co-chromatography with an authentic reference. Two minor, partly characterized carotenoids, remained unidentified. The high proportion of acetylenic carotenoids (86-90% of the total carotenoids), as previously found in Mytilus edulis (Hertzberg et al., 1988; Partali et al., 1989), is noteworthy. The presence of mytiloxanthin (12) in Harvest 1, and in higher concentration the diacetylenic alloxanthin (11) in both harvests (31-36% of total carotenoids), representing major carotenoid metabolites in Mytilus edulis (Hertzberg et al., 1988, Partali et al., 1989), suggests that similar metabolic routes (Partali et al., 1989) from microalgal fucoxanthin (17) may operate, see Table 1. Isolation of 19-hydroxy- or 19-hexanoyloxyalloxanthin from molluscs after feeding a diet containing microalgae producing 19'-hexanoyloxyfucoxanthin, such as Chrysochromulina polylepis (Bjerkeng et al., 1990), could provide evidence for this metabolic route. However, a more direct biosynthetic route to alloxanthin (11) involves dehydrogenation of zeaxanthin (6) and diatoxanthin (7), and the conversion of 5,6epoxy- or 5,6,5',6'-diepoxy-carotenoids, as proposed by Bonnett et al. (1969). In order to explain the relatively high proportion of the 4(4')-ketocarotenoids pectenolone (10) (16-50% of total carotenoid), astaxanthin (8) (4-5% of total carotenoid), 7,8-didehydroastaxanthin (9, minor), echinenone (2, minor) and 3',4'-anhydropectenolone (4), as well as the 4-ol pectenol (15, minor), another metabolic pathway involving the introduction of 4(4') oxygen functions in M. modiolus appears likely, and seems to be common to several bivalves (Miki et aL, 1982). Minor algal carotenoids such as fl,fl--carotene (1), zeaxanthin (6) and diatoxanthin (7) are plausible precursors, compatible with the chiralities assigned. The tentatively identified anhydro-zeaxanthin (3), Harvest 1, and Y,4'-anhydro-pectenolone (4), Harvest 2, are plausible metabolites of zeaxanthin (6) and diatoxanthin (7), respectively. The presence of these anhydro-carotenoids suggests a certain ability to eliminate 3(Y)-hydroxyl groups in M. modiolus, possibly reflecting the existence of a metabolic route to 3,4-dehydroretinol (vitamin A2) similar to that encountered in some freshwater fish species (Gross and Budowski, 1966). Harvest 2 differed from Harvest 1 by the presence of various C37-skeletal carotenoids (ca 5% of the
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0
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o
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~
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Carotenoids in food chains of bivalve molluscs Table 2. Carotenoids of Pecten maximus Carotenoid
fl,fl-Carotene(1) Echinenone (2) Astaxanthin (8) Pectenolone (9)
Peridinin (13) (?.is-pefidinin (13) Alloxanthin (11) Peridinol (16) Seven unidentified
% total
mg isolated
3
0.064 0.039
2 1 16
40 9 5 1 23
0.024 0.310
0.790 0.179 0.096 0.026 0.475
total carotenoids), reflecting an additional dinoflagellate diet. Thus the presence of peridinin (13, 1% of total carotenoid), the deacetylated peridinol (16, 0.1%) and the corresponding acetylenic metabolites pyrrhoxanthin (5, 0.5%), and pyrrhoxanthinol (14, 3%), support the same metabolic route of peridinin (13) as suggested for M. edulis (Partali et al., 1989). The small amount of pyrrhoxanthin (5) identified, may alternatively originate as such from dinoflagellates (Aakermann and Liaaen-Jensen, 1992). The carotenoids of Pecten maximus, a traditional seafood, will now be considered. Previous qualitative studies have revealed the presence of the diacetylenic alloxanthin (11 = pectenoxanthin = cynthiaxanthin) (Lederer, 1938; Campbell et al., 1967) and the monoacetylenic pectenolone (10) (Campbell et al., 1967), Scheme 1, from this scallop. In the present quantitative studies, see Table 2, was identified fl,fl-carotene (1, 3% of total carotenoids), and echinenone (2, 2%), astaxanthin (8, 1%) and pectenolone (10, 16%) of the common 4-keto oxidative metabolic pathway, also encountered in M. modiolus. However, the presence of high amounts of the C37-skeletal peridinin (13, 49%) and peridinol (16, 1%) pointed towards an algal diet dominated by dinoflagellates. The presence of relatively small amounts of acetylenic carotenoids is suggestive of less metabolic capacity of P. maximus to metabolize allenic to acetylenic carotenoids than for M. modiolus. Thus the presence of acetylenic C40-skeletal allenic metabolites (alloxanthin (11, 5% of the total carotenoid), and pectenolone (10, 16%)), but not acetylenic C37-skeletal carotenoids such as pyrrhoxanthin (5) and pyrrhoxanthinol (14) is possibly reflecting a lacking ability to convert C37-allenes to acetylenes in P. maximus. Seven minor carotenoids, each representing 1-4"/o of the total carotenoids, were only partly characterized. Failure to detect epoxidic C40-carotenoids such as antheraxanthin and violaxanthin, present in minor amounts in microalgae, in the investigated molluscs, may indicate lacking resorption or their rapid metabolic conversion to zeaxanthin (6) as in the gastropod Littorea littorina (Abele-Oeschger and Theede, 1991). Food chain implications
For Harvest 1 of M. modiolus, 85% of the total carotenoids may be accounted for by a diatom diet, containing fucoxanthin (17) and diatoxanthin (7) as
249
major carotenoids (Bjernland and Liaaen-Jensen, 1989). In addition C37-skeletal carotenoids derived from peridinin-producing dinoflagellates were detected in Harvest 2, accounting for some 5% of the total carotenoids. The presence of 50% of total carotenoids of C37skeletal carotenoids, for which de novo biosynthesis is restricted to the dinoflagellate algal class (LiaaenJensen, 1978; Bjernland and Liaaen-Jensen, 1989) in P. maximus, implies that also dinoflagellates are food organisms for this scallop. However, only a total of 5% of peridinin (13) and its metabolites peridinol (16), pyrrhoxanthin (5) and pyrrhoxanthinol (14) were detected in Harvest 2 of M. modiolus. The relatively high ratio between peridinin (13) and its metabolites indicates a poorer ability of P. m a x imus to convert allenic to acetylenic carotenoids than of M. modiolus. However, the level of acetylenic carotenoids may reflect the physiological status of the organisms. Thus Campbell et al. (1967) reported as much as 94% acetylenic carotenoids in P. maximus and Partali et al. (1989) isolated 47% unconverted fucoxanthin (17) of total carotenoids in one of the materials of Mytilus edulis. Bivalve molluscs are adversely affected by blooms of toxic algae, e.g. dinoflageUates (Shumway, 1990). Although M. modiolus is unaffected in some respects (Gainey and Shumway, 1988) it may remain toxic for 60 days. The presence of C37-skeletal carotenoids in molluscan seafood is therefore indicative of possible toxicity. CONCLUSION In conclusion it is inferred that M. modiolus hardly resorbs carotenes, but xantbophylls such as fucoxanthin (17), zeaxanthin (6), diatoxanthin (7) and peridinin (13) from microalgal diets. These xanthophylls are metabolized via several routes. The ability of bivalves to metabolically transform microalgal allenic carotenoids to acetylenic carotenoids (Campbell et al., 1967; Khare et al., 1973; Matsuno and Maoka, 1981; Matsuno et al., 1981, 1985; Hertzberg et al., 1988; Maoka and Matsuno, 1988; Partali et aL, 1989) seems to separate bivalves from other molluscs. The most distinct difference in carotenoid metabolism between M. modiolus and P. maximus and Mytilus edulis is the high capacity of the former two to produce 4(4')-oxygenated carotenoids, hardly encountered in M. mytilus (Khare et al., 1973, 1988; Chopra et al., 1988; Partali et al., 1989). Acknowledgements--B.B. was supported by a grant HB
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