Lipid Characteristics of Five Epinephelinae Fishes, Epinephelus

Lipid Characteristics of Five Epinephelinae Fishes, Epinephelus fasciatus, Epinephelus retouti, Cephalopholis aurantia, Cephalopholis miniatus, and Variola louti, in the Coral Reef Hiroaki Saito＊ National Research Institute of Fisheries Science, Fisheries Research Agency, 2-12-4, Fuku-ura, Kanazawa-ku, Yokohama-shi 236-8648, Japan

Abstract: The lipid and fatty acid compositions of the muscle and liver of five Epinephelinae fishes (Epinephelus fasciatus, Epinephelus retouti, Cephalopholis aurantia, Cephalopholis miniatus, and Variola louti) in the coral reef were investigated. The major polyunsaturated fatty acid (PUFA) in the Epinephelinae fish triacylglycerols was 22:6n-3 (docosahexaenoic acid; DHA), which was similar to high levels of DHA in the depot triacylglycerols of highly migratory fishes. In the phospholipids of all specimens, unusually high levels of 20:4n-6 (arachidonic acid; ARA), 22:5n-6 (docosapentaenoic acid; n-6 DPA), and 20:5n-3 (icosapen- taenoic acid; EPA) were found with markedly high levels of DHA. Noticeable levels of n-6 long-chain PUFA, such as ARA and n-6 DPA, were found in the muscle and liver polar lipids and suggested a typical profile of the fatty acid composition of coral demersal fishes. The five Epinephelinae fishes were healthful marine foods containing markedly high levels of DHA with noticeable levels of various n-3 and n-6 PUFA.

Key words: ‌arachidonic acid, coral reef, docosahexaenoic acid, Epinephelinae fishes, fish physiology, marine fish, polyunsaturated fatty acid, tropical fish

1 Introduction fishes, with growing recognition of the beneficial uses of Coral reefs form some of the most diverse ecosystems on dietary fish oils for humans7）. Compared with the many the Earth. They are home to one quarter of all marine studies on the detailed fatty acid determinations of tem- species, including fish, mollusks, crustaceans, annelids, perate and boreal fishes, little information has been avail- echinoderms, coelenterates, tunicates, and various algae. able on the detailed composition of tropical and subtropical Many studies on their ecological aspects, including biodi- fish species. Although Serranidae fishes are known as com- versity, population regulation, and fish communities, have mercially important due to their high quality, there is only been reported in the literature1, 2）; however, there are very one report on their nutritional value（s n-3 and n-6 PUFA in few reports of chemical analysis of their components. the gonads）8）. For example, despite some attention paid to Grouper（s subfamily Epinephelinae species, Serranidae, the biological and ecological aspects of Epinephelinae Percoidei, Perciformes）are important and cosmopolitan specie（s Epinephelus fasciatus3, 4）, Cephalopholis auran- food fish, the largest subfamily in the Serranidae family, tia5）, Cephalopholis miniata3－5）, and Variola louti3））, and are usually found around reefs, in tropical and sub- only a few reports on the determination of the chemical tropical waters along the coasts. Near Japan, many components have been published with a general analysis of grouper（s Epinephelinae species, Epinephelus spp.3, 4）, Cephalopholis spp.5）, and Variola spp.3））are found as sub- Abbreviations: ARA, arachidonic acid; DMA, dimethylacetals; tropical and tropical fish species, which are higher preda- DMOX, 4,4-dimethyloxazoline; DHA, docosahexaenoic acid; DPA, docosapentaenoic acid; GC/MS, gas chromatography/ tors in the coral reef and range over the southern Japanese mass spectrometry; LC, long-chain; MUFA, monounsaturated archipelago, Okinawa islands, and Ogasawara islands. fatty acids; PC, phosphatidylcholine; PE, phosphatidylethanol- There are many reports on the chemical components of amine; PUFA, polyunsaturated fatty acids; TAG, triacylglycer- pelagic seawater fishes6）, in particular, on the fatty acids of ols; TFA, total fatty acids; TL, total lipids.

＊Correspondence to: Hiroaki Saito, National Research Institute of Fisheries Science, 2-12-4 Fuku-ura, Kanazawa-ku, Yokohama 236-8648, Japan. E-mail: [email protected] Accepted January 26, 2014 (received for review December 24, 2013) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

471 H. Saito

E. fasciatus, C. aurantia, and Variola louti9）. In addition, were caught by the research vessel‘ Soyo-maru’（gross there are only a few reports presenting an analysis of the tonnage, 892 tons）, which belongs to the National Re- fatty acid composition of Epinephelinae fish species, in- search Institute of Fisheries Science, Yokohama, Japan. cluding Epinephelus malabaricus10）, Epinephelus coioi- The fish were caught at 4 different station（s 13 sample des11－13）, red grouper, Epinephelus morio14）, and hump- points of the 3 stations in the tropical sea and the 1 station back grouper, Cromileptes altivelis15）. in the subtropical sea）in the northwestern Pacific Ocean In the present study, the lipid classes and fatty acid com- from 2005 to 2008. Thirty-one individuals from five Epi- positions of five typical Epinephelinae fishes, Epinephelus nephelinae fishes were sampled. The study area extended fasciatus Forsskå（l blacktip grouper）, Epinephelus between latitudes of approximately 20° and 40° N under retouti Bleeke（r red-tipped grouper）, Cephalopholis au- two different condition（s tropical and subtropical sea）in rantia Valencienne（s golden hind）, Cephalopholis minia- the northwestern Pacific Ocean. After specimen body tus Forsskå（l Miniatus grouper, coral grouper）, and Variola length and weight were measured（Table 1）, the samples louti Forsskå（l yellow-edged lyretail）were investigated for were individually dissected, and lipids of muscle（edible their lipid characteristics as tropical demersal fishes in the part）and live（r representative of viscera）were analyzed. In coral reef. the laboratory on the vessel, each sample was immediately immersed in a solvent mixture（chloroform and methanol; 2:1, v/v）and frozen at －80℃ until extraction.

2 Materials and methods 2.2 Lipid extraction and analysis of lipid classes 2.1 Collection of sh specimens Each ordinary muscle and liver of five Epinephelinae The biological data of ﬁve grouper species, E. fasciatus, ﬁshes was individually homogenized in a mixture of chloro- E. retouti, C. aurantia, C. miniatus, and V. louti are form and methano（l 2:1, v/v）, and a portion of each homog- listed in Table 1. The E. fasciatu（s samples 1–3）, E. enized sample was extracted according to the Folch et al. retout（i samples 4–7）, C. aurantia（samples 8–9）, C. method16）. Each crude lipid was separated into classes on miniatu（s samples 10–11）, and V. lout（i samples 12–13） silicic acid column（s Merck and Co. Ltd., Kieselgel 60, were collected near Torishima Island, Ogasawara Plateau, 70-230 mesh）, and quantitative analysis of the lipid con- and Minamitorishima Island from September 2005 to De- stituents was performed using gravimetric analysis of frac- cember 2008. tions collected from column chromatography17）. The first All specimens of five Epinephelinae fishe（s Table 1）, eluate（dichloromethane/n-hexane, 2:3, v/v）was collected

Table 1 Locality of capture and biological data of the five Epinephelinae fishes.

472 J. Oleo Sci. 63, (5) 471-484 (2014) Lipid Characteristics of Five Epinephelinae Fishes, Epinephelus fasciatus, Epinephelus retouti, Cephalopholis aurantia, Cephalopholis miniatus, and Variola louti, in the Coral Reef

as steryl ester, wax ester, and diacylglyceryl ether frac- 2.4 Preparation of methyl esters and gas-liquid chroma- tions. This was followed by eluting the triacylglycerols tography（GLC）of esters （TAG）with dichloromethane and eluting the sterols with Individual components of TAG, PE, and PC fractions dichloromethane/ethe（r 35:1, v/v）; eluting the diacylglycer- were converted to fatty acid methyl esters by direct trans- ols with dichloromethane/ethe（r 9:1, v/v）; eluting the free esterification with methanol containing 1％ HCl under fatty acids with dichloromethane/methano（l 9:1, v/v）; reflux for 1.5 h, as previously reported, to produce the eluting the phosphatidylethanolamine（PE）with dichloro- fatty acid methyl esters17）. These methyl esters were puri- methane/methano（l 1:5, v/v）; eluting other minor phospho- fied using silica gel column chromatography by elution with lipids with dichloromethane/methano（l 1:20,v/v）; and dichloromethane/n-hexan（e 2/1, v/v）. eluting phosphatidylcholine（PC）with dichloromethane/ The composition of the fatty acid methyl esters was de- methano（l 1:50, v/v）17）. termined by GLC. Analysis was performed on HP-5890 Individual lipids from each lipid class, such as the phos- （Hewlett Packard Co. Ltd., Yokogawa Electric Corporation, pholipid class, were qualitatively identified with standards Tokyo, Japan）gas chromatographs equipped with an by comparing the Rf values using thin-layer chromatogra- Omegawax-250 fused silica capillary column（30 m×0.25 phy（Merck and Co. Ltd., Kieselgel 60, thickness of 0.25 mm i. d.; 0.25 μm film, Supelco Japan Co. Ltd., Tokyo, mm for analysis）. All sample lipids were dried under argon Japan）. The temperatures of the injector and the column at room temperature and stored at －40℃ under argon. were held at 230 and 215℃, respectively, and the split ratio was 1:76. Helium was used as the carrier gas at a constant 2.3 NMR spectrometry and determination of lipid classes inlet rate of 0.7 mLmin－1. Spectra were recorded on a GSX-270 NMR spectrometer Quantitation of individual components was performed by （JEOL Co. Ltd., Tokyo, Japan）in a pulsed Fourier trans- means of HP ChemStation System（A. 06 revision, Yokoga- form mode at 270 MHz in a deuterochloroform solution wa HP Co. Ltd., Tokyo, Japan）electronic integrators. using tetramethylsilane as the internal standard18）. Some fractions often contained several classes. For example, the 2.5 Preparation of 4,4-dimethyloxazoline derivatives first fraction contained wax esters, diacylglyceryl ethers, （DMOX）and analysis of DMOX by gas chromatogra- and steryl esters. The molar ratios of these classes were phy - mass spectrometry（GC/MS） determined by quantitative analysis of the NMR results The DMOX derivatives were prepared by adding an using characteristic peaks18）. The molar ratios of wax excess amount of 2-amino-2-methyl propanol to a small esters, diacylglyceryl ethers, and steryl esters were deter- amount of fatty acid methyl esters in a test tube under mined by quantitative analysis of the NMR results using argon atmosphere. The mixture was heated at 180℃ for 18 characteristic peaks. In NMR, the amount of wax esters hr17）. was obtained by the total amount of combined integrations Analysis of the DMOX derivatives was performed by a of the triplet peak（s from 3.90 to 4.20 ppm）as the two HP G1800C GCD Series I（I Hewlett Packard Co., Yokogawa protons on the ester alcohol, the diacylglyceryl ethers were Electric Corporation, Tokyo, Japan）GC/MS equipped with obtained by the singlet peak（3.50–3.80 ppm）as the two the same capillary used for determining the respective ether protons linked with glycerol carbons, and the steryl fatty acids with the HP WS（HP Kayak XA, G1701BA esters were obtained by the multiplet peak（s 4.30–4.80 version, PC workstations）. The temperatures of the injec- ppm）as one proton at the carbon linked esterified C-3 tor and the column were held at 230 and 215℃, respec- alcohol of the sterols. The actual ratios of the wax esters, tively. The split ratio was 1:76, and the ionization voltage diacylglyceryl ethers, and steryl esters in the first fraction was 70 eV. Helium was used as the carrier gas at a constant were determined as the respective integration divided by inlet rate of 0.7 mLmin－1. The fatty acid methyl esters the sum of total integrations of the three combined peaks were identified by comparing the DMOX derivative mass from 3.50 to 4.80 ppm. The actual weight of each class was spectral data obtained by GC/MS. obtained by calculating the ratio and multiplying by the total weight of the first fraction. Similarly, the third fraction 2.6 Statistical analyses sometimes contained triacylglycerol（s TAG）and sterols, Two or more experimental replication（s n＝2–4 in Table which have two characteristic peaks: for TAG（3.90–4.40 2）of lipid classes of samples were completed for each lipid ppm）, an octet-like peak for four protons, and for sterols class. For all samples of fatty acid determination by GLC, （3.40–3.60 ppm）, a multiplet for one proton. The actual more than two replication（s n＝2–8 in Table 3）were made. weight of the TAG and sterols in the third fraction was ob- Significant mean differences were determined using a tained by calculating the integration of the divided respec- one-way analysis of variance（ANOVA）. Tukey’s multiple tive peak and multiplying it by the total weight of the third procedure was used to compare the differences among fraction18）. mean values. Differences were regarded as a significance level of p＜0.05.

473 J. Oleo Sci. 63, (5) 471-484 (2014) H. Saito

Table 2 Lipid contents and classes of the five Epinephelinae fishes.

2.7 Animals the muscle neutral lipids in all specimens, with low levels All experiments were carried out according to the Japa- （roughly less than 5％ of total lipids; TL）of other neutral nese legislation regarding the welfare and management of lipids present, such as wax esters, steryl esters, diacylglyc- animals and were approved by the Bioethics Committee of eryl ethers, diacylglycerols, and free fatty acids. Steryl the National Research Institute of Fisheries Science. An esters, TAG, and sterols were the major lipids in the liver anesthetic overdose was used in all fishes. neutral lipid（s Table 2）. In the muscle and liver polar lipids of the five Epinephelinae fishes, two phospholipid（s PE and PC）were major classes with low levels of other minor phospholipids. All five Epinephelinae fish lipids mainly 3 Results contained glycerol derivative（s TAG, diacylglycerols, PE, 3.1 Lipid content and lipid classes of the ve Epinepheli- PL, and PC）and the total proportion of these derivatives nae shes reached approximately 62.2–95.9 and 64.1–89.0％ for the The total lipid contents of the five Epinephelinae fishes, muscles and livers, except for one case of liver lipid（43.3％ E. fasciatus, E. retouti, C. aurantia, C. miniatus, and V. for sample No. 11 of C. miniata）. louti samples are shown in Table 2, accompanied by the lipid classes. The lipid contents in the muscles ranged from 3.2 Fatty acid composition and its similarity in TAG de- 0.2 to 1.2％ of wet weight in all five samples, while those in posit lipids among ve Epinephelinae shes livers ranged from 2.2 to 10.9％. The fatty acids in TAG（more than 0.3％ of total fatty TAG and sterol were the major components observed in acids; TFA）in both the muscles and livers of the five Epi-

474 J. Oleo Sci. 63, (5) 471-484 (2014) Lipid Characteristics of Five Epinephelinae Fishes, Epinephelus fasciatus, Epinephelus retouti, Cephalopholis aurantia, Cephalopholis miniatus, and Variola louti, in the Coral Reef

nephelinae fish samples are shown in Tables 3–6. Although 4 Discussion the fatty acid composition of muscle TAG slightly varied 4.1 Lipid content and lipid classes of the ve Epinepheli- among the five species, the kinds of major fatty acids were nae shes almost the same in all samples tested. Six dominant fatty The lipid content of the five Epinephelinae fishes are acid（s roughly more than 3％ of TFA）in the muscle TAG listed in Table 2. The mean lipid content in the muscle were found in the samples collected from the different samples was very low, while that in the liver was high, sug- areas under varied conditions: 14:0, 16:0, and 18:0 as satu- gesting that these Epinephelinae fishes accumulate lipids rated fatty acids, 16:1n-7 and 18:1n-9 as monounsaturated primarily in the viscera, similar to other fish species18, 19）. fatty acid（s MUFA）, and 22:6n-3（docosahexaenoic acid; The lipid class composition of the five Epinephelinae fishes DHA）as n-3 PUFA. Noticeable level（s roughly more than is also shown in Table 2. TAG and sterols were the major 1％ of TFA）of eleven other fatty acids were also observed: components in muscle neutral lipids. Comparatively lower 17:0, 18:1n-7, 20:1n-9, 18:1n-7, 16:2n-4, 18:2n-6（linoleic levels of muscle TAG were found in four sample（s TAG acid）, 20:4n-6（arachidonic acid; ARA）, 22:4n-6, 22:5n-6 level: 25.7％ for sample No. 3, 18.7％ for sample No. 9, （n-6 docosapentaenoic acid; DPA）, 20:5n-3（icosapentae- 25.3％ for sample No. 11, 7.2％ for sample No. 13）, which noic acid; EPA）, and 22:5n-3（n-3 DPA）. Although the lipid are influenced by the lower lipid content（s lipid content: content in all the livers was markedly higher than those in 0.2％ for sample No. 3, 0.2％ for sample No. 9, 0.1％ for the muscles, similar dominant fatty acids in the liver TAG sample No. 11, 0.2％ for sample No. 13）. These values were also found in all samples: 14:0, 16:0, 18:0, 16:1n-7, were similar to those in other lean pelagic fishes18, 19）. This 18:1n-9, 18:1n-7, n-3 DPA（for V. louti）, and DHA with no- trend was similar to those in their liver TAG; similar lower ticeable levels of nine other fatty acid（s 17:0, 20:1n-9, TAG levels in the three sample liver lipids were observed 16:2n-4, 18:2n-6, ARA, 22:4n-6, n-6 DPA, EPA, and n-3 （38.3％ for sample No. 9 whose lipid content was 3.3％, DPA）also present. 16.4％ for sample No. 11 whose lipid content was 3.4％, and 34.9％ for sample No. 13 whose lipid content was 3.3 Fatty acid composition in tissue phospholipids in the 2.2％）. In polar lipids, PE and PC were the major compo- ve Epinephelinae shes nents in both muscle and liver, similar to those in other The major fatty acid composition of the muscle and liver pelagic fishes18－20）. For example, in the muscle lipids, phospholipids of the five Epinephelinae fishes collected medium levels of these phospholipid（s total phospholipids: from different areas in subtropical to tropical zones are 21.2–45.3％ for E. fasciatus, 5.1–30.2％ for E. retouti, also presented in Tables 3–6. The fatty acid composition in 43.0–61.4％ for C. aurantia, 38.6–61.8％ for C. miniatus, PE included low levels of dimethyl acetal（s DMA）, such as and 13.5–53.9％ for V. louti）were found. All five Epinephe- DMA 18:0 and DMA 20:1. The theoretical values of the linae lipids contained mostly glycerol derivative（s TAG, dia- fatty acid composition were obtained by subtracting the cylglycerols, PE, and PC）and the total proportion of these DMA from the TFA in PE, which resulted in the consistent derivatives reached over 60％, except in one case of the C. occurrence of the same fatty acids in the PE of organ-spe- miniata liver lipid（sample No. 11）. cific specimens of five Epinephelinae specie（s Tables 3–6）. The major seven fatty acids in PE were 16:0, 18:0, 18:1n-9, 4.2 Comparatively high levels of DHA in TAG: fatty acids ARA, n-6 DPA, EPA, and DHA with noticeable levels of in depot lipids in the ve Epinephelinae shes 18:1n-7, 22:4n-6, and n-3 DPA. Similarly, eight fatty acids The fatty acid compositions in the muscle and liver TAG were the major components in PC（Tables 3–6）: 16:0, 18:0, of the five Epinephelinae species are listed in Tables 3–6. 18:1n-9, ARA, 22:4n-6（for the livers of E. fasciatus, C. As for PUFA, only DHA was the major PUFA（＞3％）in the miniata, and V. louti）, n-6 DPA, EPA, and DHA with no- five Epinephelinae species muscle TAG with noticeable ticeable levels of 16:1n-7, 18:1n-7, 22:4n-6, and n-3 DPA. level（s ＞1％）of various n-3 and n-6 LC-PUFA, such as Although the levels of respective fatty acids in PE differed ARA, n-6 DPA, EPA, and n-3 DPA. Similar to the fatty acid from those in PC, the major PUFA in PE were quite similar composition of the muscle TAG, the major PUFA in the to those in PC; only four PUFA（ARA, n-6 DPA, EPA and liver TAG of the five Epinephelinae species were n-3 DPA

DHA）and one related long-chain（LC, C20 and C22）PUFA （for V. louti）and DHA. In spite of the neutral depot TAG （22:4n-6）were major components both in PE and PC, and whose PUFA content is generally low19, 21）, the same higher this phenomenon was also observed in the TAG in both DHA levels in both muscle and liver TAG were found in all organs. High levels of total PUFA（total PUFA; 49.8–63.6％ ﬁve Epinephelinae samples; in particular muscle TAG con- for muscle PE, 18.8–59.8％ for liver PE, 49.2–67.2％ for tained consistently medium level（s roughly more than 10％ muscle PC, 24.0–59.9％ for liver PC）in polar lipids of all of TFA）of DHA（14.8-19.4％ for the muscle of E. fas-cia- ﬁve Epinephelinae species were observed, and in particu- tus, 9.0–14.7％ for the muscle of E. retouti, 9.3–15.2％ for lar, consistently high levels of total PUFA in their muscle the muscle of C. aurantia, 12.8–16.0％ for the muscle of C. polar lipids were found（Tables 3–6）. miniatus, and 13.4–13.7％ for the muscle of V. louti）. In

475 J. Oleo Sci. 63, (5) 471-484 (2014) H. Saito

Table 3 Fatty acid composition of Epinephelus fasciatus muscle and liver examineda.

the previous papers, high levels of DHA with noticeable in these fishes, high levels of DHA were found in the TAG levels of EPA are reported in the tropical carnivorous fishes of the five Epinephelinae species. Lower EPA levels in the Caesio diagramma and Caesio tile21）, and total lipid of five Epinephelinae lipids were characteristically found Salarias patzneri22）. Similar to the fatty acid compositions （1.8–2.7％ for E. fasciatus muscle and liver TAG, 1.1–4.2％

476 J. Oleo Sci. 63, (5) 471-484 (2014) Lipid Characteristics of Five Epinephelinae Fishes, Epinephelus fasciatus, Epinephelus retouti, Cephalopholis aurantia, Cephalopholis miniatus, and Variola louti, in the Coral Reef

Table 3 Continued.

for E. retouti muscle and liver TAG, 0.7–2.6％ for C. au- other demersal fishes, such as temperate and boreal rantia muscle and liver TAG, 0.5–2.8％ for C. miniatus specie（s e.g., 7.0％ for Sparus aurata TL23）, 3.5–3.9％ for muscle and liver TAG, and 1.8–4.0％ for V. louti muscle Pagellus bogaraveo24）, 11.0–13.7％ for Gadus microceph- and liver TAG）, compared with higher levels of EPA in alus and Theragra chalcogramma25）, 9.9–17.8％ for Hip-

477 J. Oleo Sci. 63, (5) 471-484 (2014) H. Saito

Table 4 Fatty acid composition of Epinephelus retouti muscle and liver examineda.

poglossoides elassodon, Hippoglossus stenolepis, Glyp- depot TAG. This finding suggested the presence of charac- tocephalus zachirus, Lepidopsetta bi-lineata, and teristic differences between the five Epinephelinae species Limanda aspera25）, and 13.8–13.9％ for Gadus morhua TAG, and those in other demersal fishe（s sea breams23, 24）, and Hippoglossoides platessoides26））. Similar trends of cods25, 26） and flatfishes25））. the Epinephelinae TAG are seen in those of marine pelagic fishes such as highly migratory fish lipid（s DHA: 22.2– 4.3 Fatty acid composition in tissue phospholipids in the 26.0％ for TAG of Tunnus tonggol19）, 20.0–33.6％ for TL five Epinephelinae fishes: accumulations of various of Seriola dumerili, Makaira nigricans, Thunnus at- n-6 PUFA in PE and PC lanticus, Euthynnus alletteratus, Thunnus albacares27）, The major fatty acid composition of the muscle and liver and 15.5–24.7 and 13.3–19.5％ for TAG of Seriola dumer- phospholipid（s PE and PC）of the five Epinephelinae ili and Seriola rivoliana18））. Although lower levels of species are also presented in Tables 3–6. The fatty acid DHA were generally observed in the lipids of many tropical composition in PE included noticeable levels of DMA, dif- and subtropical marine fishes9, 28）, comparatively high DHA ferent from those in other common fish species except for levels in the five Epinephelinae lipids were found, even in the existence as minor components in specific organs of

478 J. Oleo Sci. 63, (5) 471-484 (2014) Lipid Characteristics of Five Epinephelinae Fishes, Epinephelus fasciatus, Epinephelus retouti, Cephalopholis aurantia, Cephalopholis miniatus, and Variola louti, in the Coral Reef

Table 4 Continued.

some fishe（s gill of catfish, Ictalurus punctatus, and tuna, the muscle PC of E. fasciatus reached 31.1–41.0％. The Thunnus alalunga29） olfactory nerve of garfish, Lepisos- higher DHA levels of the muscle polar lipids suggested the teus osseus, and brains of goldfish, Carassius auratus30））. concentration of DHA in their tissue polar lipids27, 31）. These As for PUFA in the polar lipids, markedly high levels of findings suggested a similarity of the accumulation mecha- DHA and high levels of various n-3 and n-6 LC-PUFA, such nism of high DHA between the Epinephelinae species and as ARA, n-6 DPA, and EPA with noticeable levels of other marine fishes, such as highly migratory fishes19－21, 27）. 22:4n-6 and n-3 DPA, were found in the muscle and liver In contrast, the high levels of n-6 PUFA, such as ARA PE of the five Epinephelinae species. Similar to the major and n-6 DPA, in the Epinephelinae polar lipids were unusu- PUFA in the muscle PE, the same PUFA were observed in ally characteristic, and the profile of PUFA in their polar the muscle PC of all samples. Compared with lower levels lipids markedly differed from those in other marine fishes, of DHA in the Epinephelinae species TAG, higher DHA such as most marine carnivorous fishes6－7, 19）, which have levels in the polar lipids of the Epinephelinae species were low or trace levels of n-6 LC-PUFA. With respect to the observed. For example, numerical DHA percentages of the level of PUFA, almost all marine fishes in temperate and muscle PE of E. fasciatus were 35.2–38.2％ and those for boreal seas mainly contain n-3 PUFA, while ARA is gener-

479 J. Oleo Sci. 63, (5) 471-484 (2014) H. Saito

Table 5 Fatty acid composition of Cephalopholis aurantia and Cephalopholis miniata muscle and liver examineda.

ally undetectable or negligible in their lipids6）. Similar to （Siganus canaliculatus21））in the coral reef. The lipids of noticeable levels of ARA in other tropical fish polar and Epinephelinae species, which are highest predators in the gonadal lipid（s 2.7–3.2％ for C. diagramma PE and PC coral reef, might be influenced by their prey, which are and 2.9–3.3％ for C. tile PE and PC21）, 2.3–4.3％ for T. small young and juvenile herbivorous fishes, such as Sigani- tonggol PE and PC19）, and for gonadal lipids of 24 tropical dae fishes. Consequently, high levels of both n-3 and n-6 fish species8））, a high n-6 LC-PUFA（ARA and n-6 DPA） LC-PUFA were observed in the tropical fish lipids in the found simultaneously in all polar lipids of the Epinepheli- coral reef, while high levels of only n-3 PUFA with trace nae species might be characteristic of tropical fish lipids at levels of n-6 PUFA were found in the northern fish lipids. the coral reef. Compared with the very low levels of n-6 Although the function of n-6 PUFA in marine organisms LC-PUFA in the northern（temperate and boreal）species, has not yet been clearly demonstrated, the Epinephelinae higher levels of ARA were found in the herbivorous fishes species may accumulate ARA similar to the accumulation

480 J. Oleo Sci. 63, (5) 471-484 (2014) Lipid Characteristics of Five Epinephelinae Fishes, Epinephelus fasciatus, Epinephelus retouti, Cephalopholis aurantia, Cephalopholis miniatus, and Variola louti, in the Coral Reef

Table 5 Continued.

mechanism of n-3 PUFA32）. Differing from the essentiality n-6 LC-PUFA and, more specifically, that various n-6 PUFA of n-3 PUFA for marine fishes32）, the Epinephelinae species and DHA were the characteristic fatty acids in the tissue accumulate n-6 PUFA as a possible substitute for n-3 PE and PC of the Epinephelinae fishes. This finding also PUFA in order to maintain cell membrane fluidity. Further- indicated that tropical demersal Epinephelinae fishes are more, these n-6 LC-PUFA might be important for repro- healthful marine foods rich in DHA with various n-3 and duction for the tropical fish species because high levels of n-6 LC-PUFA. Many Epinephelinae fishes are important n-6 LC-PUFA in gonadal lipids are often observed in many food fish, and some of them are now aquacultured; tropical fish species8）, similar to ARA essentiality for the however, V. louti often has toxic compounds. Establish- larvae of marine fishes33－35）. The present study confirmed ments of farming technology of these useful fishes may that the five Epinephelinae fish polar lipids contained solve the problem and open a possibility of utilizing these markedly high levels of DHA with unusually high levels of tropical species, such as V. louti.

481 J. Oleo Sci. 63, (5) 471-484 (2014) H. Saito

Table 6 Fatty acid composition of Variola louti muscle and liver examineda.

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