Department of Food Science and Technology, Tokyo University of Marine Science and Technology (4-5-7, Konan, Minato-ku, Tokyo , Japan) 3

Transcription

1 Journal of Oleo Science Copyright 2013 by Japan Oil Chemists Society Actual Ratios of Triacylglycerol Positional Isomers and Enantiomers Comprising Saturated Fatty Acids and Highly Unsaturated Fatty Acids in Fishes and Marine Mammals Toshiharu Nagai 1, Yumiko Matsumoto 2, Yanying Jiang 2, Keiko Ishikawa 2, Tokuhisa Wakatabe 2, Hoyo Mizobe 1, Kazuaki Yoshinaga 1, Koichi Kojima 1, Ikuma Kuroda 3, Tadao Saito 4, Fumiaki Beppu 2 and Naohiro Gotoh 2* 1 Tsukishima Foods Industry Co. Ltd. (3-17-9, Higashi Kasai, Edogawa-ku, Tokyo , Japan) 2 Department of Food Science and Technology, Tokyo University of Marine Science and Technology (4-5-7, Konan, Minato-ku, Tokyo , Japan) 3 GL Sciences Inc. (6-22-1, Nishi Shinjuku, Shinjuku-ku, Tokyo , Japan) 4 Graduate School of Agricultural Science, Tohoku University (1-1, Tsutsumi-dori, Amamiya-machi, Aoba-ku, Sendai , Japan) Abstract: It has been previously shown that the positional isomers of triacylglycerol (TAG) containing palmitic acid (P) and highly unsaturated fatty acids (HUFAs) such as DHA (D) and EPA (E) vary between fishes and marine mammals. However, it has not yet been understood why in marine mammals HUFAs are located only at the α position when two palmitic acid chains combine, and not in fishes. In order to gain further understanding of the biosynthetic pathways involved in the formation of these asymmetric TAGs, we investigated whether the HUFA in the TAG of marine mammals exists predominantly at the sn-1 or sn-3 position. We examined the TAG positional isomers and enantiomers in marine organisms in detail. As a result, while PDP and PEP were not detected, sn-ppd and sn-ppe were found in abundance in marine mammals. For fishes, on the other hand, PDP, PEP, sn-ppd, and sn-ppe were all identified. In the case of TAGs that contain two HUFAs and one palmitic acid, marine mammals were rich in DPD and EPE whereas fishes were rich in sn-pdd and sn-pee. Key words: chiral column, enantiomer, fish, highly unsaturated fatty acid, marine mammal, triacylglycerol 1 INTRODUCTION Triacylglycerol TAG, consisting of one glycerol and three fatty acids, is a major component of lipids 1. Many fatty acid species exist in nature; therefore, the possible number of different combinations of three fatty acids in TAG is enormous. In order to take this variation into account, the term TAG molecular species is used. There are two possible positions at which the fatty acids bind to the glycerol: at the primary alcohols α position and the secondary alcohol β position 2. These two positions can be distinguished by pancreatic lipase in the digestion process, with only the fatty acids located at the α positions being hydrolyzed 3. In the case of a TAG that consists of two types of fatty acid, A and B, with two As and one B located on glycerol backbone, there are two possible TAG isomers; B can either be located at an α position AAB or the β position ABA. As these TAGs consist of the same types and numbers of fatty acids, they are termed TAG positional isomers. The carbon atom connected to the secondary alcohol becomes a stereogenic center in AABtype TAGs. In the Fischer projection of such species Table Abbreviations: APCI, atmospheric pressure chemical ionization; C, caprylic acid; CSP, chiral stationary phase; D, docosahexaenoic acid (DHA); DAG, diacylglycerol; E, eicosapentaenoic acid (EPA); HPLC, high-performance liquid chromatography; HUFA, highly unsaturated fatty acid; MAG, monoacylglycerol; MRM, multiple reaction monitoring; MS, mass spectrometry; O, oleic acid; P, palmitic acid; PL, phospholipid; Rf, rate of flow; SIR, single ion recording; TAG, triacylglycerol; TLC, thin layer chromatography. * Correspondence to: Naohiro Gotoh, Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo , Japan ngotoh@kaiyodai.ac.jp Accepted September 2, 2013 (received for review August 6, 2013) Journal of Oleo Science ISSN print / ISSN online

2 T. Nagai, Y. Matsumoto, Y. Jiang et al. Table 1 Structures of TAG isomers used in this study. sn-ppd: 1,2-dipalmitoyl-3-docosahexaenoyl-sn-glycerol rac-ppd: 1,2-dipalmitoyl-3-docosahexaenoyl-rac-glycerol PEP: 1,3-dipalmitoyl-2-eicosapentaenoyl-sn-glycerol sn-ddp: 1,2-didocosahexaenoyl-3-palmitoyl-sn-glycerol rac-ddp: 1,2-didocosahexaenoyl-3-palmitoyl-rac-glycerol EPE: 1,3-dieicosapentaenoyl-2-palmitoyl-sn-glycerol PDP: 1,3-dipalmitoyl-2-docosahexaenoyl-sn-glycerol sn-ppe: 1,2-dipalmitoyl-3-eicosapentaenoyl-sn-glycerol rac-ppe: 1,2-dipalmitoyl-3-eicosapentaenoyl-rac-glycerol DPD: 1,3-didocosahexaenoyl-2-palmitoyl-sn-glycerol sn-eep: 1,2-dieicosapentaenoyl-3-palmitoyl-sn-glycerol rac-eep: 1,2-dieicosapentaenoyl-3-palmitoyl-rac-glycerol 1, if the fatty acid esterified with the secondary alcohol was located on the left-hand side and the hydrogen bound to the asymmetric carbon was located on the right-hand side, the upper esterified position is called the sn-1 position, the esterified position on the secondary alcohol is called the sn-2 position, and the remaining position is called the sn-3 position 2. Accordingly, the TAG positional isomer, AAB, contains two different TAG enantiomers: sn-aab 1,2-diA-3-B-sn-glycerol and sn-baa 1-B-2,3-diAsn-glycerol. Although there is no difference in physical properties between a pair of TAG enantiomers, some organisms or enzymes can recognize only one of the two structures. For example, it has been reported that lingual lipase selectively hydrolyzes short- and medium-chain fatty acids located at the sn-3 position in rodent milk fat 4, 5. A method for determining the fatty acid distributions at the sn-1, 2, and 3 positions of natural TAGs was reported by Brockerhoff in This technique was used to successfully reveal the distribution of fatty acids at each sn position in plant oil, animal fat, milk fat, fish oil, and hen egg. For example, uneven distributions of highly unsaturated fatty acids HUFAs at the sn-2 position of TAG in fish oil and at the sn-1 and 3 positions in seal oil were reported 6. However, intact molecular species of TAG positional isomers and enantiomers were not separated in this work. Itabashi and Takagi reported the resolution of enantiomeric monoacylglycerol MAG and diacylglycerol DAG on chiral high-performance liquid chromatography HPLC and applied their methods for the stereospecific analysis of TAG from edible oils and fats 7, 8. Yet, their methods required partial hydrolysis of the acyl groups of TAG, and precolumn derivatization of DAG to 3,5-dinitrophenylurethanes before performing chiral HPLC analysis. Iwasaki et al. directly resolved some TAG enantiomers using chiral HPLC on a polysaccharide-based chiral stationary phase without prior derivatization. However, this is only possible if the TAG enantiomers comprise very different acyl groups 9. Of the many examples of chiral HPLC analyses of glycerolipids reviewed by Itabashi in , only Iwasaki s report showed separation of TAG enantiomers that were maintained intact. Nevertheless, this particular method could not separate the major TAG molecular species having acyl residues with C16 C18-long chain lengths that are contained in edible oils and fats. We recently developed a method for resolving some racemic asymmetric TAGs i.e., 1,2-dioleoyl-3-palmitoyl-rac-glycerol rac-oop and 1,2-dipalmitoyl-3-linoleoyl-rac-glycerol rac-ppl into pairs of enantiomers, in combination with a recycle HPLC system equipped with a chiral column, using methanol as the mobile phase 11. This method did not require any derivatization before analysis. Recently, Lisa and Holcapek reported an excellent method for the separation of TAG regioisomers positional isomers and enantiomers by chiral HPLC with gradient elution of hexane and 2-propanol

3 TAG ENANTIOMER IN MARINE ORGANISMS However, it was difficult to accurately measure the ratios of regioisomers and enantiomers in hazelnut oil and human plasma samples because while many of these were simultaneously separated, some of them overlapped. In our previous report, reversed-phase HPLC was adopted for the fractionation of asymmetric TAGs such as OOP in palm oil, prior to chiral separation of sn-oop and sn-poo. To quantify TAG positional isomers and enantiomers in natural lipids, another separation mode such as reversed-phase or silver-ion HPLC should be combined with chiral HPLC. We recently reported the actual ratio of TAG positional isomers in marine organisms consisting of HUFAs such as DHA and EPA and saturated fatty acids such as palmitic acid. These were analyzed using HPLC-atmospheric pressure chemical ionization APCI /mass spectrometry MS equipped with a polymeric ODS column 13. We assumed that the actual ratios of the TAG positional isomers consisting of palmitic acid, EPA, and DHA partially represented the characteristics of TAGs bound to HUFA in marine organisms. As a result, we revealed that almost all HUFAs were located at the α position in the case of TAGs with one HUFA and two palmitic acids in marine mammals; however, there was more variation in the location of HUFA in fishes. However, it has not yet been understood why in marine mammals HUFA is located only at the α position of TAG when two palmitic acids are present. To gain further understanding of the biosynthetic pathway of these asymmetric TAGs, more detailed analysis of TAG enantiomers is required. Therefore, in the present study, we applied our developed method 11 for the separation of TAG positional isomers and enantiomers of marine organisms to confirm whether HUFA in the TAGs of marine mammals exists predominantly at the sn-1 or sn-3 position. 2 MATERIALS AND METHODS 2.1 Chemicals and materials TAG isomers shown in Table 1 were in-house products. All other reagents were purchased from Wako Pure Chemical Industries, Ltd. Osaka, Japan. Fish meat was bought at a supermarket in Tokyo, Japan. Capsules of harp seal oil were obtained from a health food store in Tokyo, Japan. Steller s sea lion meat was obtained by a mail order from a shop in Tatsuno, Japan. 2.2 Lipid extraction and fractionation of TAG The lipid samples were extracted from the fish or Steller s sea lion meat by the Bligh and Dyer procedure 14. The extracted lipids, along with the harp seal oil from the capsules, were separately dissolved in hexane and spotted on a silica gel plate to separate the TAG fraction by thin layer chromatography TLC. The spotted TLC plate was developed with a petroleum ether/diethyl ether/acetic acid 80:20:1, v/v/v mixture. The TAG was recovered by scraping off the band which showed the same rete of flow Rf value as an authentic sample of TAG such as triolein, and extracting with a chloroform/methanol 20:1, v/v mixture. The extracted TAG sample was dried by rotary evaporation under vacuum, weighed, dissolved in 2-propanol, and stored in an argon-purged screw-capped vial at 40 until analysis. 2.3 Partial purification of DPD/DDP and EPE/EEP from TAG samples Because the amounts of DPD/DDP and EPE/EEP were very small, it was necessary to perform their partial purification before determining the TAG positional isomers by LC/MS Section 2.4. The purified TAG samples were first adjusted to 1 with 2-propanol and then chromatographed. The peak, including DPD/DDP and EPE/EEP, which eluted at 11 min, was fractionated data not shown using semipreparative HPLC consisting of a pump LC- 10AD, Shimadzu Corporation, Kyoto, Japan, a UV-Vis detector SPD-10A, Shimadzu Corporation at 215 nm, an injection valve equipped with a 200-μL sample loop Model 7725, Rheodyne LLC, Rohnert Park, CA, and an ODS column Inertsil ODS, 250 mm 10 mm i.d., 10 μm, GL Sciences Inc., Tokyo, Japan. A mixture of acetonitrile and 2-propanol 70:30, v/v was used as the mobile phase. The flow rate and injection volume were 3.0 ml/min and 100 μl, respectively. The column temperature was 25. The retention time for collection was confirmed using reference standards. The fraction containing DPD/DDP and EPE/ EEP was concentrated and adjusted to 1000 μg/ml. 2.4 Quantification of TAG positional isomers Eight types of TAG isomers, rac-ppd, PDP, rac-ppe, PEP, rac-pdd, DPD, rac-pee, and EPE, were selected as standards. All the TAG isomers were mixed and dissolved in 2-propanol. The final concentrations of respective TAG isomers were adjusted to 1000, 500, 250, 100, or 50 μg/ml and used to obtain calibration curves using the single ion recording SIR mode of LC/MS, the setup and conditions of which were as follows: a pump PU-611C, GL Sciences Inc., a UV-Vis detector UV-702, GL Sciences Inc., a column oven MO-706, GL Sciences Inc., an injection valve equipped with a 50-μL sample loop Model 7725, Rheodyne LLC, an MS with an APCI probe ZMD, Waters Corporation, Milford, MA, and tandem jointed ODS columns Inertsil ODS-P, 250 mm 4.6 mm, i.d. 5 μm, GL Sciences Inc.. The column temperature, mobile phase, flow rate, and injection volume were 18, acetonitrile/2-propanol/ hexane 3:2:1, v/v/v, 0.8 ml/min, and 20 μl, respectively, for the resolution of PDP/PPD or PEP/PPE; and 22, acetonitrile/2-propanol 3:2, v/v, 0.8 ml/min, and 20 μl, respectively, for the resolution of DPD/PDD or EPE/PEE. The capillary voltage, cone voltage, source block tempera- 1011

4 T. Nagai, Y. Matsumoto, Y. Jiang et al. ture, desolvation temperature, and desolvation gas flow rate of the APCI ion source conditions were 3.5 kv, 40 V, 135, 400, and 500 L/h, respectively. The ions for analyses by SIR mode were as follows: m/z 871, 897, 917, and 969 corresponding to PEP/PPE, PDP/PPD, EPE/PEE, and DPD/PDD, respectively, which were ammonium-adduct ions of TAGs. The TAG sample solutions obtained in Sections 2.2 and 2.3 were analyzed in the same way, and each analysis was performed in triplicate. The obtained peak areas in the chromatograms were used to calculate the actual ratios of TAG positional isomers according to a previously reported method Fractionation of asymmetric TAGs from TAG samples of marine organisms Collection of the asymmetric TAG positional isomers from purified TAG samples was carried out by the method described in Section 2.4. The retention times were confirmed using standard TAGs. Each TAG sample was adjusted to approximately 1 mg/ml, and 20 μl of the sample was chromatographed on Inertsil ODS-P. Each peak of PPD, PPE, DDP, and EEP was confirmed by SIR chromatograms, and the samples were collected. The fractionated solution was dried under a nitrogen stream, and the residue was weighed and dissolved in 2-propanol to a concentration of approximately 100 μg/ml. 2.6 TAG enantiomer separation by recycle HPLC with chiral column The asymmetric TAG fractions obtained in Section 2.5 were then separated into enantiomers. The setup and analytical conditions are described below. The recycle HPLC system composed of a recycle pump PU712R, GL Sciences Inc., an autosampler GL-7420, GL Sciences Inc., a column oven CO-705C, GL Sciences Inc., a UV-Vis detector UV702, GL Sciences Inc., and two automatic valves VALVE UNIT 401, FLOM Co., Ltd., Tokyo, Japan to allow the analytes to pass through the same column repeatedly and the extra-column volume of the autosampler was reduced using a sample loop during the recycle run. An operational software EZChrom Elite, Agilent Technologies, Inc., Santa Clara, CA was used to control the recycle HPLC system. An APCI/MS Quattro micro API, Waters Corporation and its operational software MassLynx Ver. 4.1, Waters Corporation was used as a detector in combination with the recycle HPLC system. A chiral column CHIRAL- CEL OD-RH, 4.6 mm i.d. 150 mm, 5 μm, Daicel Corporation, Tokyo, Japan and a guard cartridge CHIRALCEL OD-RH, 4.0 mm i.d. 10 mm, 5 μm, Daicel Corporation were used for the resolution of sn-ppd/sn-dpp, sn-ddp/ sn-pdd, and sn-ppe/sn-epp. Similarly, another chiral column CHIRALCEL OZ-3R, 4.6 mm i.d. 150 mm, 3 μm, Daicel Corporation and a guard cartridge CHIRALCEL OZ-3R, 4.0 mm i.d. 10 mm, 3 μm, Daicel Corporation were employed for the resolution of sn-eep/sn-pee. The column temperature was 25. Methanol 100 was used as the mobile phase for both columns, with a flow rate was 0.5 ml/min. The TAG standard was dissolved in 2-propanol and adjusted to 1 mg/ml. A 10 μl aliquot of the standard solution was injected from the autosampler and the target TAG enantiomers having passed through the columns and UV-Vis detector were returned to the entrance of the recycle pump by a recycle valve. After the target TAG enantiomers were sufficiently resolved, they were forced into the MS through the recycle valve, which changed the outlet flow pass of the UV-Vis detector from the entrance of the recycle pump to the APCI probe of the MS. The parameters of the APCI/MS were as follows: ionization mode, APCI positive; corona current, 3.0 μa; source temperature, 120 ; desolvation temperature, 450 ; cone gas flow, 50 L/h; desolvation gas flow, 200 L/h; data acquisition mode, MRM. The precursor-to-product ion MRM transitions for sn-ppd/sn-dpp, sn-ppe/sn-epp, sn-ddp/sn-pdd, and sn-eep/sn-pee were m/z , , , and , respectively. The collected AAB-type TAG solutions were analyzed using the LC/MS system; each analysis was carried out thrice. The resolution and elution order of the AAB-type TAG enantiomers comprising DHA and palmitic acid, or EPA and palmitic acid, were first confirmed using the standard compounds, sn-ppd, rac-ppd, sn-ppe, rac-ppe, sn-ddp, rac-ddp, sn-eep, and rac-eep. The averages of the TAG enantiomer ratios were directly calculated using the peak area ratios of the MRM chromatograms. 3 RESULTS AND DISCUSSION All racemic TAGs required recycle runs to achieve enantiomer resolution. For the sufficient resolution of rac-ppd, rac-ppe, and sn-ddp into the corresponding enantiomers, the racemic TAG should be run five times through the column. The elution order was found to be the same as observed in our previous report 11, with the TAG enantiomer with the unsaturated fatty acid bound at the sn-1 position eluting earlier than the other enantiomer. The chiral stationary phase CSP of CHIRALCEL OD-RH was cellulose tris 3,5-dimethylphenylcarbamate coated on silica gel, which is known for its excellent versatility 15. However, good resolution was not achieved for rac-eep using this material. CSPs consisting of polysaccharide-based phenylcarbamate derivatives have a helical structure with many chiral sites at equal intervals, and can recognize chiral analytes. A well-known mechanism by which chiral recognition occurs is the three-point attachment model 16, 17. In this model, at least three configuration-dependent attractive contact points between the chiral receptor and the chiral 1012

5 TAG ENANTIOMER IN MARINE ORGANISMS substrate are necessary. In the case of asymmetric TAGs, the difference in the fatty acid moieties between the sn-1 and 3 positions would be recognized by the CSP, with some of the asymmetric TAGs able to separate into the corresponding enantiomers. In this study, rac-ppe was separated into its enantiomers using CHIRALCEL OD-RH; however, rac-eep could not be separated by this method, even though both TAGs had palmitic acid and EPA moieties at the sn-1 and 3 positions. In contrast, this behavior was not observed in the chiral separation of rac-ppd and rac- DDP. These results suggest that an EPA moiety located at the sn-2 position adversely affects the chiral separation of rac-eep, and the conformation of CHIRALCEL OD-RH could not recognize the structure of sn-eep or sn-pee. As an alternative, another chiral column, CHIRALCEL OZ-3R, the CSP of which is cellulose tris 3-chloro-4-methylphenylcarbamate coated on silica gel, was employed for the resolution of rac-eep after screening different columns. Interestingly, good resolution was achieved by using this column; however, the enantiomer with the unsaturated fatty acid moiety bound at the sn-3 position was eluted earlier Fig. 1, which was opposite to the results found for CHIRALCEL OD-RH. The functional groups bound to the phenyl group of CHIRALCEL OD-RH are two methyl Fig. 1 MRM chromatograms of TAG enantiomers in sardine. Resolution of A) sn-ppd and sn-dpp, B) sn-ddp and sn-pdd, C) sn-ppe and sn-epp, and D) sn-eep and sn-pee. Column: CHIRALCEL OD-RH (4.6 mm i.d. 150 mm, 5 μm) with a guard cartridge for the resolution of (A) sn-ppd/sn-dpp, (B) sn-ddp/sn-pdd, and (C) sn-ppe/sn-epp, CHIRALCEL OZ-3R (4.6 mm i.d. 150 mm, 3 μm) with a guard cartridge for the resolution of (D) sn-eep/sn-pee. Column temperature: 25 ; mobile phase: methanol 100%; flow rate: 0.5 ml/min; number of passage through column: five times by recycle HPLC system; MS conditions: ion source: APCI positive; MRM transitions for m/z 880 > 625 for sn-ppd/sn-dpp, m/z 854 > 597 for sn-ppe/sn-epp, m/z 952 > 625 for sn-ddp/sn-pdd, and m/z 900 > 597 for sn-eep/sn-pee. 1013

6 T. Nagai, Y. Matsumoto, Y. Jiang et al. Table 2 Actual ratios of TAG-positional isomers and TAG-enantionmers consisted of two palmitic acids and one HUFA in marine mammals and fishes (%). Marine Mammals Fishes Steller Sea Lion Harp Seal Skipjack Tuna Sardine sn-dpp / PDP / sn-ppd 13.6 / 0 / / 0 / / 43.8 / / 64.9 / 26.5 sn-epp / PEP / sn-ppe 0 / 0 / / 0 / / 26.6 / / 49.0 / 32.9 (Average Value, n = 3) Table 3 Actual ratios of TAG-positional and TAG-enantionmers consisted of one palmitic acid and two HUFAs in marine mammals and fishes (%). Marine Mammals Fishes Steller Sea Lion Harp Seal Skipjack Tuna Sardine sn-ddp / DPD / sn-pdd 0 / 89.2 / / 70.5 / / 5.9 / / 6.6 / 79.9 sn-eep / EPE / sn-pee 0 / 84.1 / / 77.8 / / 17.3 / / 17.8 / 72.4 (Average Value, n = 3) groups, which possess electron-donating characteristics. On the other hand, the functional groups bound to the phenyl group of CHIRALCEL OZ-3R are one methyl group and one chloride. As the chloride is electron-withdrawing, the electron density on the phenyl group might affect the order of elution of the TAG enantiomers. Iwasaki et al. reported that the order of elution of 1-eicosapentanoyl-2,3- dicapryloyl-sn-glycerol sn-ecc and 1,2-dicapryloyl- 3-eicosapentanoyl-sn-glycerol sn-cce was reversed by changing the chiral column to that containing chloride 9. Similar to the present study, the column with the chloride group resulted in the earlier elution of sn-cce in comparison to sn-ecc. Substituents such as chloride affect the electron density of the phenyl groups of the polysaccharide-based chiral column, which is likely to be responsible for the alterations in the order in which the TAG enantiomers are eluted. The contents of TAG isomers in the samples were quantified using a calibration curve constructed using TAG standards. The actual ratios of sn-ppd/pdp/sn-dpp, sn-ppe/pep/sn-epp, sn-ddp/dpd/sn-pdd, and sn-eep/ EPE/sn-PEE in marine organisms are summarized in Tables 2 and 3. As has been previously reported 13, marine mammals do not contain PDP and PEP, which is in contrast to fishes species, which contain TAGs with HUFA bound at the sn-2 position. However, the trends in the actual ratios of sn-ppd/sn-dpp and sn-ppe/sn-epp in marine mammals and fishes are the same, with HUFA mainly bound at the sn-3 position. TAGs that accumulate in organisms are mainly biosynthesized via the glycerol-3-phosphate pathway 18, 19. In this pathway, sn-glycerol-3-phosphate formed from glycerol is esterified with fatty acid acyl- CoA at the sn-1 position by sn-glycerol-3-phosphate acyltransferase. The formed 1-acyl-sn-glycerol-3-phosphate is further esterified with fatty acid at the sn-2 position by 1-acyl-sn-glycerol-3-phosphate acyltransferase. The formed 1,2-diacyl-sn-glycerol-3-phosphate phosphatidate is converted to 1,2-diacyl-sn-glycerol sn-1,2-dag by phosphatidate phosphohydrolase, which is then esterified with fatty acid acyl-coa at the sn-3 position by diacylglycerol acyltransferase to form TAG. This biosynthetic pathway means that the respective fatty acids are esterified at three different positions in the TAG individually rather than simultaneously. Phospholipids PL are also synthesized via the same pathway 20, and the fatty acids bound at the sn-1 and 2 positions of PL are esterified by the same acyltransferase. It is likely that 1-acyl-sn-glycerol-3-phosphate acyltransferase in marine mammals does not have a preference for HUFA as a reaction substrate. In contrast, diacylglycerol acyltransferase in marine mammals is more similar to the enzymes found in fishes, resulting in HUFAs binding at the sn-3 position of TAG. The fatty acid species bound at the sn-2 position are extremely important for maintaining the homeostasis of organisms. Phospholipase A 2 hydrolyzes fatty acids bound at the sn-2 position of PL in the cell membrane 21. The resulting free fatty acid is used for the formation of eicosanoids such as prostaglandins and leukotrienes, which are local hormones. These substances are mainly synthesized from arachidonic acid C20:4n-6 or EPA C20:5n-3, and HUFA must be located at the sn-2 position of PL. However, in marine mammals, HUFA does not exist at the sn-2 position of TAG. As indicated above, the binding of fatty acids at the sn-2 position of TAGs and PLs is controlled by the same acyltransferase; therefore, it would be interesting to investigate the fatty acid composition at the sn-2 position of PLs in marine mammals. Experiments to this end are our next target, and the results will be reported in due course. 1014

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