FISHERIES AND MARINE SERVICE. Translation Series No. 3350

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1 FISHERIES AND MARINE SERVICE ARCHIVES Translation Series No Composition of the esterified fatty acids of astaxanthin diester in the skin of seven red fishes by Nobuo Tsukuda,-and Tadashi Kitahara Original title: Sekishoku gyorui gyohichu ni okeru esuteru gata astaxanthin no ketsugo shibosan ni tsuite From: Tokaiku Suisan Kenkyusho Kenkyu Hokoku, (77): 89-95, 1974 Translated by the Translation Bureau(YS/PS) Multilingual Services Division Department of the Secretary of State:of. Canada Department of the Environment Fisheries and Marine Service Halifax Laboratory Halifax, N.S pages typescript

2 4. e DEPARTMENT OF THE SECRETARY OF STATE G4> SECRÉTARIAT D'ÉTAT TRANSLATION BUREAU MULTILINGUAL SERVICES DIVISION e&s eheureï CANADA BUREAU DES TRADUCTIONS DIVISION DES SERVICES MULTILINGUES TRANSLATED FROM - TRADUCTION DE Japanese AUTHOR - AUTEUR INTO EN English FrA1 4.e Lrô TSUKUDA,Nobuo and KiTAHARA,Ta.dashi TITLE IN ENGLISH - TITRE ANGLAIS Composition of the Esterified Fatty Acids of Astaxanthin Diester in the Skin of Seven Red Fishes TITLE IN FOREIGN LANGUAGE (TRANSLITERATE FOREIGN CHARACTERS) TITRE EN LANGUE ÉTRANGÉRE (TRANSCRIRE EN CARACTÈRES ROMAINS) Sekishoku gyorui gyohichu ni okeru esuteru gata astaxanthin no ketsugo shibosan ni tsuite. REFERENCE IN FOREIGN LANGUAGE (NAME OF BOOK OR PUBLICATION) IN FULL. TRANSLITERATE FOREIGN CHARACTERS. RÉFÉRENCE EN LANGUE ÉTRANGÉRE (NOM DU LIVRE OU PUBLICATION), AU COMPLET, TRANSCRIRE EN CARACTÈRES ROMAINS. Tokai kensui ho. REFERENCE IN ENGLISH - RÉFÉRENCE EN ANGLAIS Bull. TokeReg. Fish. Res. Lab. PUBLISHER ÉDITEUR PLACE OF PUBLICATION LIEU DE PUBLICATION Not given. Japan YEAR ANNÉE 1974 DATE OF PUBLICATION DATE DE PUBLICATION VOLUME ISSUE NO. NUMÉRO 7? PAGE NUMBERS IN ORIGINAL NUMÉROS DES PAGES DANS L'ORI GINAL PP NUMBER OF TYPED PAGES NOMBRE DE PAGES DACTYLOGRAPHIÉES 14 REQUESTING DEPARTMENT MINISTÈRE.CLIENT Environment TRANSLATION BUREAU NO. NOTRE DOSSIER N 0 7G 4/7.2. BRANCH OR DIVISION TRANSLATOR (INITIALS) Fisheries Service DIRECTION OU DIVISION TRADUCTEUR (INITIALES) YS / PS PERSON REQUESTING Allan T. Reid DEMANDÉ PAR FEB YOUR NUMBER VOTRE DOSSIER N 0 DATE OF REQUEST DATE DE LA DEMANDE UNeDITED TRANSLATION e'of information only TRADUCTION NON REVISEF Informtion seulement (REV.2a0)

3 D&PARTMENT OF THE SECRETARY OF STATE TRANSLATION BUREAU, MULTILINGUAL SERVICES D VISION SECRÉTARIAT D'ÉTAT BUREAU DES TRADUCTIONS. DIVISION DES SERVICES MULTILINGUES CLIENT'S NO. DEPARTMENT. DIVISION/BRANCH CITY N 0 DU CLIENT MINISTRE DIVISION/DIRECTION VILLE BUREAU NO. N DU BUREAU.. Environment Fisheries Service Ottawa,ont, LANGUAGE LANGUE JaDanese YS/pS. TRANSLATOR (INITIALS) TRADUCTEUR (INITIALES) FEB Composition of the Esterified Fatty Acids of Astaxanthin Diester in the Skin of Seven Red Fishes Authore TSUKUDA,Nobuo and KITAEARA, Tadashi* Name of publication: Bull. Tokai Ileg. Fish. Res. Lab. No. 77 'Place and date of publication: Japan. March, Abstra -et: Studies were conducted on the compositions of the esterified fatty acids of astaxanthin diester which were separated and purified from the skin of seven red fishes of marine origin by the method of column chromatography. The fatty acids of astaxanthin diester were identified by thin-layer chromatography and gas liquid chromatography using the preparations of fatty acids. The main components of fatty acids were oleic acid and palmitic acid. The sum of these two fatty acids reach -ed 70 to 90% of the total fatty acids in seven fishes. Myrisiic acid, palmitoleic acid, stearic acid and linoleic acid seemed to exist in sniall amounts as other fatty acids. Accordingly, it is regarded that the astaxanthin diester in the skin of red fishes mainly combine with oleic acid and palmitic acid at the 3, 3'-positions of astaxanthin. tful 0 >, 4 E,e) w c 0 4LZ,.170 F- r; z o 0 It is said carotinoid pigment in the skin of P. 8 9 fishes is present for the most part as ester. For example, Hirao and others 1 ' 2) say that in the case of Received on November 7, Tokai-ku Fisheries Research C Laboratory's contributions: A-532. z D te. Otqru University of Commerce. 3 home, 5-21, Midori, Otaru Shi, 047, ob..à 00-d 0..31

4 2 goldfish(carassius auratus) 90% and likewise in the case of killifish(oryzias latips) 95% of allcarotinoid is considered to be 1n ester form. The writers 3) have also noted that in all cases of gurnard(chelidonichthys kumu), akamutsu(d8derleiria berycoides), yumekasago(helicolerus bilgendorfi) between 90 and 95% of the entire carotinoid is in the ester form. However, there have hardly been any reports regarding.the substance combined with ester of the esterified carotinoid. The only report that can be found is by Karrer and others 4) stating that the epiphasic astaxanthin(3;3, - dihydroxy-4,4 1 -diketo-b-carotene) in lobster (Astacusgammarus) is astaxanthin dipalmitate but the writers have not come across any studies on the composition of esterified carotinoid in fish skin. An attempt has been made to study the substance combined with ester of the esterified astaxanthin taken from the skin of seven'red fishes from the Japanese home waters. The results are discussed in this paper. Materials for Experiments and Method p.90 'Sample fishes The following seven fishes were used: Fresh alfonsins(beryx splendens), usumebaru(sebastes thompsoni), Kichiji(Sebastolobus macrochir), gurnards(chelidonichthys kumu), chikamekintoki (Priacanthus boops) purchased from the Tokyo Central Wholesale Market, and kasago (Sebastiscus marmoratus), vumekasaw. in2iluim5.2 hilendorfi) purchased immediately after landing. at the fish market of Choshl City in Chiba Prefecture.

5 3 Nlethod: Colored tissue was obtained from the skin and fin of the sample fishes to which was added sulfuric anhydride sodium. After dehydrating it, pigment was extracted several times with acetone. Whole oil was obtained from the concentrate of acetone extract solution. From the whole oil, esterified astaxanthin was purified by chromatography. This ester combination was decomposed by saponification. The: obtained. fatty acids and its methylated products were identified by means of thin-layer chromatography and gas chromatography. Experiments and the Results Separation and purification of the esterified astaxanthin. The whole oil obtained from the sample fishes contains large amount of oils and fats;therefore, 100m0. of acetone was first added to the whole oil, and was left in a refrigerator at the temperature of -l0 C overnight, and the white lipid which was educed from the whole oil was sifted.out. The dark red filtrate obtained was converted by solving to petroleum ether, and chromatography was carried out by using silicic acid magnesium(woelm, degree of activation 1)as adsorbent. The volume of acetone contained in the petroleum ether was gradually increased, developed and eluted. Elution took place in the following manner. Before the volume of acetone

6 4 contained in the petroleum ether reached 10%,most of the lipids and of the yellow carotinold were eluted. The principal part of the red carotinoid was eluted by the petroleum ether containing 20'ic, acetone. This dark red fraction was concentrated and was made into petroleum ether solution, in addition, chromatography by aluminium (Merck, standard article, degree of activation was carried out once, chromatography by silicagel(merck, degree of activation was carried out twice. As it was stated above, the volume of acetone contained in the petroleum ether was gradually increased, expanded, and eluted. Each time, the main portion of the red carotinoid eluted by petroleum ether containing 3% acetone wascollected and the impueities were removed as much as possible. The results of the preliminary experiments showed that if more chromatography was carried out, the yield of red carotinoid would not only drop noticeably, but also the extinction coefficient of the principal fraction would not increase as much as expected. On the contrary, in some cases the extinction coefficient of the principal fraction decreased a little. Therefore, purification by chromatography was carried out four times in total, and not more. Finally, the absorption spectrum of the dark red fraction which was obtained was a. curve with a single

7 5 maximum absorption at 4, 72mp in petroleum ether(b.p c). Also, this dark red fraction elves a single spot by thin-layer chromatoeraphy with silicagel G. Moreover, adsorbability and elution ability on the chromato-column, as well as the division by petroleum ether and 95% methanol, agree very well with the astaxanthin diester in the carotinoid of the sea crayfish(panulirus japonicus) Which Matsuno and others 5) pointed out. For this reason, this sample is regarded as astaxanthin diester (Fie. 1) o OR' Fig. 1. Astaxanthin diester 1% The yield and the extinction coefficient(e lcm ) of the astaxanthin diester obtained from the sample fishes are shown'in Table 1. Table 1 shows that, there is a ' difference amongst the extinction coefficients of the astaxanthin diester obtained from the seven fishes; and moreover, when the extinction coefficient of astaxanthin dioleate is sought b Y calculation based on the extinction coefficient ) fthe astaxanthin the value should be about 1,100 in n-hexane. However, all the extinction coefficients of the samples obtained here are Jr than this value. that the purity of Based on these bearines, it seems sample astaxanthin diester differs somewh9t dependinp: on the kinds of fishes, and all of them

8 6 still containa until amount of sterol and other lipid. Table 1. Yields and extinction coefficient (E lice'm ) of astaxanthin die.ster in the skin of seven red fishes. Japanese common name ; Amount Yeilds of 1% Weight Latin name of S astaxanof wholse ou rce thin E I sample Date of "" oil 472mp in fishes of fises.. I(English common name) n diester z-hexane purcnase mg / i g Kin medai 1 Beryx splendens i (a alfonsin) ,040 3, 200 Tokyo* Nov. 28, '72 Usumebaru!Sebastes thompsoni ,900 Tokyo* Dec. 12, '72 I (a rockfish) Kichiji!Sebastolobus macrochir I (Channel rockfish) , 000 Tokyo* Feb. 21, ' b6 _ IChelidonichthys arlicituvratt.rurva eevristr, kumu 1(a gurnard) , 800 Tokyo* Feb. 28, '73 Kasago Sebastiscus marmoratus (a rockfish) , 600 Choshi** July 2, '73 Yumekasago, Helicolemts hilgendorfi (a rockfish) ,900 Choshi** July 2, '73 Chikame- ;Priacanthus boops ,900 Tokyo* July 10, '73 kintoki (a bigeye) i * Tokyo central wholesale market ** Choshi fishing port -1 Production of fatty acids and of fatty acids methyl esters : All Astaxanthin diesters obtained from the seven fisheswere saponified at room temperature by the customary method in nitroeen gas, in a dark place, and after removing the non-saponified matter by ether extraction, the rest of the soap solution was turned into acetic acid acidity; the fatty acids were separated and extracted by ether. In this casqa considerable amount of carotinoid (astasin) was still dissolved in the ether. extraction solution, and since carotinoid(astasin) interferes with identification of the fatty acids, carotinoid was removed by chromatography with silicagel(nerck, degree of

9 activation That is to say, a chromatography tube of lcm bore was packed with silicageon a length of 3 cm. When a petroleum ether mixture solution of carotinoid and fatty acids was adsorbed on it, all the fatty acids were eluted with petroleum ether containing 20% ether;however, it was confirmed that hardly any carotinoid was eluted. Therefore with tbis method, the interfering carotinoid was removed and fatty acids were obtained. Fatty acids methyl ester was produced and obtained by 5% hydrochloric acid methanol method from the fatty acids which were obtained by the above mentioned method. Identification of the fatty acids 1) Identification by thin-layer chromatography(tlc). TLC was carried out on the fatty acids of the above Mentioned seven specimens prepared from astaxanthin diester. TLC was carried out by, first of all, immersing silicagel F 254(Merck) platesin petroleum ether containing 10%tetradecaneand then havine them ' dried by air, tben using reversed phase plates. The development solvent was a mixture solution(tetradecaresaturation) of acetic acid and acetonitrile with 1:1 ratio, and the condition of development was 3 hours at a temperature of 30 0, and the detection was made by 60% sulfuric acid color development method.

10 As it is shown in Fig. 2 according to the results of TLC the principal spots agree with standard oleic acid; therefore, the *nain component of all of them is presumed to be oleic acid. However as far as the other fatty acids are concerned, identification was difficult due to tailing Moreover with this method,the mutual separation of the so-called critical partner fatty acids such as for example,oleic acid and palmitic acid, is said to be p 92 0 Li A 0 0 os 0 a 0 I 4 a T^^ :r: ^ Fig. 2. Reversed-phase partition thin-layer chromatography of fatty acids obtained from the astaxanthin diester in seven red fishes. Plate: Tetradecane-impregnated silica get F254 (Merck) Solvent system: Acetic acid-acetonitrile, 1/1 (tetradecane saturation) Developing time: 180 min (301C) Indicator: 60 ô aqueous sulfuric acid A: Authentic saturated fatty acids S) stearic acid, P) palmitic acid, M) myristic acid, L) lauric acid B: Authentic unsaturated fatty acids. O) oleic acid, Li) linoleic acid, Ln) linolenic acid 1: Kinmedai, Bcryx splcndens 2: Usucnebaru, Se bnsle:c tkompsani 3: Kichiji, Sebastolnbus macroclrir 4: Ilabb, cfrelldanlclydlys ku» ut 5: Kasaga, Sebastiscus nrnrnmrntus 6: â u: c'- aago, Hclicoleiurs hilgendorfi 7: Chikamekintoki, Frincanrthras boops

11 9 difficult. Therefore, with this method even though the presence of the oleic acid was co.nfirmed, the composition of the fatty acids could not be defined in all samples. The above results were the same when TLC was carried out with the fatty acid methyl esters ' of the seven specimens. 2) Identification by gas chromatography(glc). p.92 GLC was carried out on the fatty acids methyl esters of the above mentioned seven specimens,using a Hitachi's Model K53 apparatus underthe following conditions: retention phase, polyester FA, 2m, 3mm the temperature of the column at 200 C,-injection inlet teinperature 26eC, carrier gas(nitrogen) flux 4o ml/min., and 10/ti of ether solution of the sample was injected. As it is shown in Fig. 3-9 the results of the GLC show similar patterns in all the cases. That is to say, the greatest peak is oleic acid and the next greatest peak is palmitic acid. The totalvalume of these two fatty acids di 1fem3depending upon the sample but in all of them the total voluine is between 70 and 90%. With the rest of the fatty acids, the preserce of the following was confirmed: myristic acid, palmitoleic acid, stearic acid and linoleic acid. In addition, several kinds of unknown fatty acids in very small quantities were present.

12 10 are A 5 10 Retention time, min Fig. 3. Kinmedai, Beryx shlendens Retention time, min Fig. 4. Usumebaru, &bastes thompsom : ; Retention time, min Retention time, min Fig. 5. Kiehiji, Sebastolobus macrorhir Fig. G. llôbô, Chelidonichthys blunt

13 Retention time, min. Fig. 7. Kasago, Scbastiscus marmoratus Retention time, mi n Fig. 8. Yumekasago, Helicolenus hilgendorfi Figs Gas chromatograms of fatty acid methyl esters obtained from the astaxanthin diester in seven red fishes. Dotted line is authentic fatty acid methyl esters. From the left peaks, methyl laurate, methyl myristate, methyl palmitate, methyl stearate, methyl oleate, methyl linoleate and methyl linolenate Retention time, min Fig. 9. Chikainekintoki, Priacanthus boops

14 12 Discussion It has already been pointed out that Karrer and others 4) state that the composition of esterified fatty p.95 acids of astaxanthin diester found in animal tissue, is in the case of a lobster, dipalmitate. However, according to our present experiment the composition of the esterified fatty acids of astaxanthin diester of the seven red fishes differs distinctly from the views * expressed by Karrer and others. That is to say, according to the results of TLC and GLC, the esterified fatty acids combined with astaxanthin in the sample fishes cannot be regarded as. jlaving a simple composition, but several kinds of fatty acids are presumed to have a part in it. Tbse fatty acids are for the most part C 16 - C and unsaturated fatty acids ; and the greatest quantity among the principal component is oleic acid and the next greatest is palmitic acid. Besides these, the presence of myristic acid, palmitoleic acid, stearic acid, and linoleic a.cid were equally noted, and moreover,trace quantities of several kinds of fatty acids were detected. However, when the purity of astaxanthin diester of the sample fishes WRS soureht from the extinction coefficient, even though its purity differed somewhat depending upon the molecular weight of the combined fatty

15 ' acids, when this was assumed to be astaxanthin dioleate, the degree of purity reached approximately between 75 and ', and it still contained between 5 to 2 52: of impurities. Therefore it is important to note the effect of the fatty acids which are derived from the impurities. Based on these facts, the very small peak in GLC could be regarded as deriving from the impurities. However, judging from the GLC patterns which are common to the seven specimens, it is probably correct to say that as far as the ester compound fatty acids are concerned the principal components are oleic acid and palmitic acid. That is to say, generally, astaxanthin diester in red fishes, is assumed to combine of mainly oleic acid and palmitic acid at the 3,3' position of the astaxanthin molecule. Besides these, although small in quantity, there is a strong possibility that the following have a part in the ester combination; myristic acid, palmitoleic acid, stearic acid and linoleic acid. However it is difficult to arrive at an accurate conclusion from the results of this experiment alone. Research in greater detail will follow in the future, Summary Astaxanthin diester which is the main pigment in the skin of seven fishes, was conected. With these samples the composition of the esterified fatty acids were examined by TLC and GLC.

16 14 The results showed that the principal components of the fatty acids were oleic acid and paimitic acid and the rest of the fatty acids only amounted to very small quantity. Therefore it was presumed that the esterified astaxanthin in the skin of red fishes combined mainly with oleic acid and palmitic acid at the 3,3' position of the astaxanthin molecule. The writers are grateful to Mr. K. NIiwa and Mr. Iida of the utilization section of Tokai Ku Fisheries Research Laboratory for their great assistance in the sample analysis by GLC. Bibliography: 1) Hirao, S,, Kikuchi B., Taguchi, P:issuishi,(Bulletin of the Japanese Soc. of Scientific Fisheries), 29.; (1963). 2) Hirao, S., Kikuchi, R., and Hama, T., Bull. Jap. Sci, Fish. 35, (1969). 3) Tsukuda Y., Bull. -Tokai Rea. Fish. I3es. Lab. 70, (1972). 4) Karrer, Pop Loewe, Los und Hiibner, H. : Helv. Chim. Acta, 18, 96-l00 -.(1935) S). Matsuno T., Kusunoto, T,, Watanabe, T., Ishihara, Y., ntissuishi,(bulletin of the Japanese Soc. of Scientific Fisheries) 39, 43-50(1973). 6) Kanemitsu, T., Aoe, H., Nissuishi, (Bulletin of the Japanese Soc. of Scientific Fisheries) 24, (195$).

FISHERIES AND MARINE SERVICE. Translation Series No Investigation of the lipid composition of several species of fish

FISHERIES AND MARINE SERVICE. Translation Series No Investigation of the lipid composition of several species of fish FISHERIES AND MARINE SERVICE ARCHIVES Translation Series No. 3637 Investigation of the lipid composition of several species of fish by A.G. Rudenko, and V.I. Lisovskaya Original title: Issledovanie sostava