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A simple screening method for discrimination between commercial extra virgin olive oils and their blends with other vegetable oils was developed. Squalene, which was contained relatively high amounts in virgin olive oil, was determined by HPLC after a simple pretreatment that was carried out by dilution of oil samples with 2-propanol. Tyrosol, which was contained at relatively high concentration in virgin olive oil among phenolic compounds, was determined by HPLC after a simple liquid-liquid extraction. When using squalene and tyrosol contents as axes, extra virgin olive oils could be discriminated from pure olive oils, blended oils (extra virgin olive oils with sunflower oil or grapeseed oil) and other vegetable oils. These results suggest that determining squalene and tyrosol in seed oil samples could be useful in distinguishing between extra virgin olive oil and blended oils as a screening method.

Fatty acids in marine algae have attracted the attention of natural chemists because of their biological activity. The fatty acid compositions of the Solieriaceae families (Rhodophyceae, Gaigartinales) provide interesting information that unusual cyclic fatty acids have been occasionally found. A survey was conducted to profile the characteristic fatty acid composition of the red alga Solieria pacifica (Yamada) Yoshida using gas chromatography-mass spectrometry (GC-MS), infrared spectroscopy (IR), and proton nuclear magnetic resonance spectroscopy (1H-NMR). In S. pacifica, two cyclopentyl fatty acids, 11-cyclopentylundecanoic acid (7.0%), and 13-cyclopentyltridecanoic acid (4.9%), and a cyclopropane fatty acid, cis-11,12-methylene-hexadecanoic acid (7.9%) contributed significantly to the overall fatty acid profile. In particular, this cyclopropane fatty acid has been primarily found in bacteria, rumen microorganisms or foods of animal origin, and has not previously been found in any other algae. In addition, this alga contains a significant amount of the monoenoic acid cis-11-hexadecenoic acid (9.0%). Therefore, cis-11,12-methylene-hexadecanoic acid in S. pacifica was likely produced by methylene addition to cis-11-hexadecenoic acid.

Palm oil and lard are edible fats which are rich in palmitic (P) and oleic acids (O). In this study, triacylglycerol (TAG) positional isomers (symmetric and asymmetric isomers) and enantiomers (asymmetric isomers) in palm oil and lard were quantified simultaneously by using liquid chromatography/mass spectrometry. The CHIRALPAK IF-3 column used in our previous study recognized the difference of TAG isomers consisting of P and O in palm oil and lard, separated sn-OPP/sn-PPO/sn-POP and sn-OPO/sn-OOP/sn-POO into each isomer peak, and enabled the quantification of these TAG isomers with good recovery (95&ndash, 120%). Although sn-POP and sn-OPO were the major TAGs in palm oil and lard, a comparison of the abundance ratios of TAG enantiomers such as sn-PPO/sn-OPP and sn-OOP/sn-POO revealed that there were slightly more TAG enantiomers with O at the sn-1 position and P at the sn-3 position in palm oil and P at the sn-1 position and O at the sn-3 position in lard. These results were consistent with previous reports for the positional distribution of fatty acids of palm oil and lard. This is the first study that has enabled all TAG isomers consisting of P and O in natural oils and fats to be individually quantified by mass spectrometry.

The beneficial health effects of edible sea urchin consumption in mice fed a normal (ND) or high-fat diet (HFD) were investigated in this study. Notably, sea urchin-administered (250, 500, 1000 mg/kg) mice exhibited lower body, liver, and visceral fat weights, lower plasma levels of aspartate aminotransferase and alanine aminotransferase, and lower hepatic triacylglycerol levels than those fed carboxymethyl cellulose (CMC). Despite the high levels of cholesterol found in sea urchins, intake of these organisms had no effect on plasma cholesterol levels among the mice tested. Additionally, sea urchin consumption resulted in enhanced levels of arachidonic acid and docosahexaenoic acid within mouse livers. Lastly, mice fed HFD with sea urchin (500 mg/kg) exhibited increased mRNA expression of uncoupling protein-1 within brown adipose tissue, compared with those fed HFD with CMC. In conclusion, consumption of sea urchin might provide a protective effect against the development of obesity and/or nonalcoholic fatty liver disease.

Using high-performance liquid chromatography coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOF-MS), we have developed a new method for detection and identification of furan fatty acids (F-acids), which are widely distributed in living organisms and foods as minor lipid components and are known to have antioxidant and anti-inflammatory effects. For this purpose, total fatty acids prepared from the testis lipids of Japanese chum salmon (Oncorhynchus keta) were examined without any concentration or isolation of F-acids. In negative ESI mode, F-acids gave a prominent [M−H]− ion, by which individual F-acids could be detected and identified. High-resolution extracted ion chromatograms clearly showed the occurrence of five major F-acid homologs as already reported by GC/MS. The method was successfully applied to several fish samples and revealed the occurrence of F-acids for the first time in the two New Zealand fish, hoki (Macruronus novaezelandiae) and school shark (Galeorhinus galeus).

The rapid and simultaneous separation of triacylglycerol (TAG) enantiomers and positional isomers was achieved using chiral high performance liquid chromatography (HPLC). TAGs composed of two fatty acids, which were both saturated (P: palmitic acid or S: stearic acid) and unsaturated (O: oleic acid or L: linoleic acid; e.g., sn-PPO/sn-OPP/sn-POP: 1,2-dipalmitoyl-3-oleoyl-sn-glycerol/1-oleoyl-2,3-dipalmitoyl-sn-glycerol/1,3-dilpalmitoyl-2-oleoylglycerol), were resolved into three peaks using CHIRALPAK IF-3 without recycling on the HPLC system. For example, the mixture of sn-PPO/sn-OPP/sn-POP was resolved in 30 min, although it took 150 min to resolve sn-PPO/sn-OPP using CHIRALCEL OD-RH in a previous study using a recycling HPLC system. This novel chiral HPLC method was applicable for the separation of other TAG isomers, including sn-OOP/sn-POO/sn-OPO, sn-PPL/sn-LPP/sn-PLP, sn-LLP/sn-PLL/sn-LPL, sn-SSO/sn-OSS/sn-SOS, sn-OOS/sn-SOO/sn-OSO, sn-SSL/sn-LSS/sn-SLS, and sn-LLS/sn-SLL/sn-LSL. For TAGs composed of three fatty acids containing both saturated and unsaturated fatty acids, the POL isomers were not sufficiently separated but the PSO and SOL isomers were partially separated into several peaks. Their elution order could be estimated by the fragment ions generated in the ion source of the mass spectrometer. However, TAGs consisting of only saturated or unsaturated fatty acids (e.g., sn-PSP/sn-PPS/sn-SPP and sn-OLO/sn-OOL/sn-LOO) were not separated. This novel chiral HPLC method is especially applicable for the analysis of TAG composition of semi-solid fats such as palm oil.

The environmental stresses that sessile organisms experience usually fluctuate dramatically and are often recurrent. Terrestrial plants can acquire memory of exposure to sublethal heat stress to acquire thermotolerance and survive subsequent lethal high-temperature stress; however, little is known concerning whether seaweeds acquire thermotolerance via heat-stress memory. We have demonstrated that the red seaweed Bangia fuscopurpurea can indeed acquire memory of sublethal high-temperature stress, resulting in the acquisition of thermotolerance that protects against subsequent lethal high-temperature stress. Moreover, the maintenance of heat-stress memory was associated with a slight increase in the saturation level of membrane fatty acids. This suggests that the modification of membrane fluidity via changes in membrane fatty acid composition is involved in the establishment and maintenance of heat-stress memory in B. fuscopurpurea. These findings provide insights into the physiological survival and growth strategies of sessile red seaweeds to cope with recurrent changes in environmental conditions.

The red seaweed G. vermiculophylla is rich in polyunsaturated fatty acids with 20 carbon atoms, mainly arachidonic acid (AA) and eicosapentaenoic acid, which are precursors of prostaglandins (PGs). The present study aimed to elucidate the capability of the seaweed in releasing PGs using acetone powder as the crude enzyme. Crude enzyme was prepared using cold acetone. The crude enzyme was incubated with AA at different concentrations (0.1– 4 mg). For determination of PG contents, 5 µL of sample as the test solution corresponding to 0.2 g wet mass of the seaweed was injected into the HPLC. For mass spectrometer analysis, an HPLC system connected with mass spectrometer was used. Results of the study showed that t he released PGs from incubation of acetone powder and AA analyzed by HPLC consisted of PGE 2 , 15-keto-PGE 2 , 15-hydroperoxy-PGE 2 , PGA 2 , and AA while PGs detected by LC-MS were PGF 2α , PGE 2 , 15-keto-PGE 2 , 15-hydroperoxy-PGE 2 , and PGA 2 . The capability of the red algae in producing PGs was affected by available oxygen, aspirin, a cyclooxygenase inhibitor, and AA concentration. The crude enzyme of the red alga (250 mg) was capable to produce 1.63 µg and 1.32 µg of PG 2 and 15-keto-PGE 2 from incubation with 0.25 mg of AA. This method could be the one way to provide PGs in vitro to fulfill demands of PGs in the pharmaceutical industry.

The red macroalga Agarophyton chilensis is a well-known producer of eicosanoids such as hydroxyeicosatetraenoic acids, but the alga produces almost no prostaglandins, unlike the closely related A. vermiculophyllum. This indicates that the related two algae would have different enzyme systems or substrate composition. To carry out more in-depth discussions on the metabolic pathway of eicosanoids between the two algae, we investigated the characteristics of glycerolipids, which are the substrates of eicosanoids production, of A. chilensis and compared them to the reported values of A. vermiculophyllum. In A. chilensis, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylcholine (PC) were the major lipid classes and accounted for 44.4% of the total lipid extract. The predominant fatty acids were arachidonic acid (20:4n-6), an eicosanoids precursor, and palmitic acid (16:0). The 20:4n-6 content was extremely high in MGDG and PC (>70%), and the 16:0 content was extremely high in DGDG and SQDG (>40%). A chiral-phase HPLC analysis showed that fatty acids were esterified at the sn-1 and sn-2 positions of those lipids. The glycerolipid molecular species were determined by reversed-phase HPLC–ESI–MS analysis. The main glycerolipid molecular species were 20:4n-6/20:4n-6 (sn-1/sn-2) for MGDG (63.8%) and PC (48.2%), 20:4n-6/16:0 for DGDG (71.1%) and SQDG (29.4%). These lipid characteristics of A. chilensis were almost the same as those of A. vermiculophyllum. Hence, the differences of the eicosanoids producing ability between the two algae would not be due to the difference of substrate composition but the difference of enzyme system.

In the filamentous brown alga Ectocarpus siliculosus, male and female sex is expressed during the haploid parthenosporophyte phase of the life cycle. Here, we found that male parthenosporophytes displayed thermotolerance whereas female specimens displayed severely reduced viability at 25 °C and 28 °C. Profiling of polyunsaturated fatty acids showed that n-3 and n-6 were the predominant species in male and female parthenosporophytes, respectively, and that the n-3/n-6 fatty acid ratio was not affected by a temperature change. Both male and female parthenosporophytes contained the sterols fucosterol, cholesterol, and ergosterol, but these were present at higher levels at 10–25 °C in female specimens than in males. Thus, these fatty acids and sterols would be expected to make the membranes more rigid in the female compared to the male, which is opposite to the paradigm that increased rigidity confers thermotolerance. Our results suggest that the sex-dependent thermotolerance in E. siliculosus parthenosporophytes is not explained by the relationship between membrane fluidity and differences in fatty acids and sterol compositions.

γ-Stearidonic acid, 18:4n-6, a potential product of β-oxidation of arachidonic acid (20:4n-6), was only recently positively identified in a living organism—a thermophilic cyanobacterium Tolypothrix sp., albeit at low levels, whilst some indirect evidence suggests its wider presence, e.g. in a unicellular marine alga. We have prepared 18:4n-6 using an iodolactonisation chain-shortening approach from 22:5n-6 and obtained its 1H-, 13C-, COSY- and HSQC NMR spectra, with 18:5n-3 spectra also recorded for a comparison. The GC and GC-MS behaviour of its methyl ester was also studied. Like another Δ3 polyunsaturated acid, octadecapentaenoic (18:5n-3), 18:4n-6 rapidly yields 2-trans isomer upon formation of dimethyloxazoline derivative. On a polar ionic liquid phase (SLB-IL100, 200 °C) the methyl ester could be mistaken for 18:3n-3, while on methylsilicone phase (BP1, 210 °C) it eluted ahead of 18:3n-6 and 18:4n-3, suggesting that when present it may be easily misidentified during GC analysis of fatty acids.

When hairless NCN24 mice with atopic dermatitis (AD) were sprayed with a petroleum-containing alkaline salt spring water rich in metaboric acid and sodium bicarbonate, AD symptoms diminished. Reversed-phase HPLC with fluorescence detection (HPLC/FD) and online MS revealed that fatty acid (FA) composition of the skin surface lipids was similar to that in non-AD mice compared with that in AD mice. Strong negative correlations were noted between the levels of total serum immunoglobulin E (IgE) and palmitoleic acid and between the levels of total serum IgE and branched-hexadecanoic acid. Conversely, a strong positive correlation was noted between the levels of total serum IgE and linoleic acid. The present study demonstrates that the petroleum-containing spring water alters the FA composition of skin surface lipids in AD mice, which can be used as an index to evaluate inflammation.

The red alga Gracilaria vermiculophylla is a well-known producer of prostaglandins, such as PGE2 and PGF2α. In this study, the characteristics of glycerolipids as substrates of prostaglandin production were clarified, and the lipid classes, fatty acid composition, and glycerolipid molecular species were investigated in detail. The major lipid classes were monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and sulfoquinovosyldiacylglycerol (SQDG), as well as phosphatidylcholine (PC), which accounted for 43.0% of the total lipid profile. Arachidonic acid (20:4n-6), a prostaglandin precursor, and palmitic acid (16:0) were the predominant fatty acids in the total lipid profile. The 20:4n-6 content was significantly high in MGDG and PC (more than 60%), and the 16:0 content was significantly high in DGDG and SQDG (more than 50%). Chiral-phase high-performance liquid chromatography determined that fatty acids were esterified at the sn-1 and sn-2 positions of those lipids. The main glycerolipid molecular species were 20:4n-6/20:4n-6 (sn-1/sn-2) for MGDG (56.5%) and PC (40.0%), and 20:4n-6/16:0 for DGDG (75.4%) and SQDG (58.4%). Thus, it was considered that the glycerolipid molecular species containing one or two 20:4n-6 were the major substrates for prostaglandin production in G. vermiculophylla.

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl-γ-cycodextrin (HP-γ-CD)-modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate-15 mM borate buffer (pH 9.0) containing 30 mM HP-γ-CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)- and (R)-enantiomers of regioisomeric 8-, 11-, 12-, and 15-HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8- and 12-HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8-HETE was found to be a mixture of 98% (R)-enantiomer and 2% (S)-enantiomer, while the 12-HETE was a mixture of 98% (S)-enantiomer and 2% (R)-enantiomer. The present study demonstrates that the HP-γ-CD-modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs. This article is protected by copyright. All rights reserved

Detailed fatty acid compositions of five species of the brown algae Sargassum (S. fulvellum, S. horneri, S. boreale, S. thunbergii, and S. yezoense) were determined using silver ion solid phase extraction, gas chromatography (GC), and GC-mass spectrometry (GC-MS) techniques. In addition to a high number of typical saturated and unsaturated fatty acids, the GC-MS spectra of the 4,4-dimethyloxazoline derivatives of fatty acids revealed the occurrence of small amounts of unusual non-methylene-interrupted (NMI) fatty acids with Δ5 unsaturation, namely, 5,9-eicosadienoic (5,9-20:2), 5,11,14-eicosatrienoic (5,11,14-20:3), and 5,11,14,17-eicosatetraenoic (5,11,14,17-20:4) acids. Of these three NMI acids, the 5,9-20:2 acid was found to be the most abundant (0.4%-2.3% of the total fatty acids) and was detected for the first time in algae.

繁殖地を離れて1週間にもおよぶロングトリップ中にミズナギドリ類は餌の大部分を消化・吸収し，吸収しづらいトリアシルグリセロール（TAG）やワックスエステルを胃油として胃に蓄積する．胃油のもととなる餌生物を特定するため，胃油中のTAGの脂肪酸組成を新潟県粟島で育雛中のオオミズナギドリにおいて分析し，潜在的な餌の脂肪酸組成と比較した．胃油の脂肪酸組成は，カタクチイワシあるいはサンマに似ていたが，他の外洋性の生物を食べた可能性も完全には否定できない．この2種だけを食べたと仮定すると，その比率はオイルベースでカタクチイワシが77％，サンマが24％と推定された．

Since the enantiomer resolution of synthetic mono- and diacylglycerols as 3,5-dinitrophenylurethanes was achieved by chiral-phase high-performance liquid chromatography (HPLC) in the 1980s, the methodology has been extended to the resolution of various synthetic and naturally occurring chiral glycerolipids. The recent development of polysaccharide-based chiral stationary phases along with optimized chromatographic elution conditions has permitted direct enantiomer resolution of mono-, di-, and triacylglycerols without previous derivatization. However, there are still some compounds for which enantiomer resolution is difficult to achieve, such as phosphatidylcholine and phosphatidylethanolamine. With the development of new derivatization reagents and chromatographic techniques, resolution of these difficult compounds may be possible in the near future. Chiral-phase HPLC will become considerably important in the future as a valuable tool for further expansion of the field of lipidomics and a better understanding of lipid metabolism.

The lipid and fatty acid compositions in two edible subtropical algae (the brown alga Cladosiphon okamuranus Tokida and the green alga Caulerpa lentillifera J. Agardh) were determined to clarify their lipid characteristics and nutritional values. Glycolipids and phospholipids were the major lipid classes, with significant levels of triacylglycerols. Polyunsaturated fatty acids (PUFA) were the major fatty acids of both algae. The lipid class composition and major fatty acids were similar in both the algal species, irrespective of wild and cultured specimens. Typical n-6 PUFA, such as 18:2n-6 (linoleic acid) and 20:4n-6 (arachidonic acid), occurred in characteristically high levels in both of the algae. High levels of n-3 PUFA were measured in all lipid classes of both species without 22:6n-3 (docosahexaenoic acid), 18:3n-3, 18:4n-3, and 20:5n-3 (eicosapentaenoic acid) for Cl. okamuranus; and 16:3n-3, 18:3n-3, and 20:5n-3 for Ca. lentillifera. The finding suggests that the green algal species, which mainly biosynthesizes short-chain (C16 and C18) PUFA, differs from that of the brown alga, which is capable of biosynthesizing high 20:5n-3 levels. The PUFA levels in glycolipids of the two algal species comprised up to 60%, even though they are subtropical marine species. High n-6 PUFA levels in the algal lipids probably influence the significant levels of n-6 PUFA in herbivorous fishes, because the n-6 PUFA levels in marine fish lipids are generally undetectable or negligible.

Photosensitivity was observed in the mice used in bioassays of diarrhetic shellfish poisoning. The mice were intraperitoneally injected with the extracts from the midgut glands of cultured scallops Patinopecten yessoensis gathered in early spring in Funka Bay, Hokkaido, Japan. The injections induced atypical symptoms in the mice within 24 h. The symptoms included piloerection, substantial eye discharge, swelling of the ears and head, and ear scratching. They were similar to those reported in studies of photosensitivity caused by pyropheophorbide in the midgut glands of abalones, which are herbivorous gastropods. The problematic scallops also accumulated a large amount of pyropheophorbide-a. The amount determined with high-performance liquid chromatography was 300–530 μg per 1 g of the homogenate of the midgut glands. The dose of pyropheophorbide sufficient to cause photosensitivity in a mouse is estimated to be 0.99–2.3 mg per mouse with 20-g body weight. Moreover, comparative tests in mice showed that the onset of the symptoms required light and a substantial amount of pyropheophorbide. Therefore, we determined that the observed symptoms were photosensitivity caused by pyropheophorbide. Scallops, which are suspension-feeding bivalves, can accumulate a large amount of pyropheophorbide in the midgut glands depending on the habitat environment.

エゾノネジモクに存在するジガラクトシルジアシルグリセロール（DGDG）の分子種を明らかにするために，DGDG からジアシルグリセロールを調製し，これをジニトロフェニルウレタン誘導体に変換して HPLC と MS で分析した。キラル HPLC では sn-1,2-ジアシル型であることが明らかになり，逆相 HPLC では 16 種の分子種が明瞭に分離された。逆相 HPLC/MS で得られた [RCOO]- イオンを用いて各分子種が同定された。主要分子種は 20:5n-3/18:4n-3 と 20:5n-3/18:3n-3 であった。

A glycerolipid acyl-hydrolase was purified 19-fold with a yield of 11% from the prostaglandin-producing red alga Gracilaria vermiculophylla by ammonium sulfate precipitation, anion-exchange chromatoraphy and gel filtration chromatography. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of the final preparation showed a single band corresponding to a molecular mass of 20 kDa, but Superdex 200 fast protein liquid chromatography exhibited a molecular mass of 40 kDa. Accordingly, it was suggested that the purified enzyme was a homodimer of a 20 kDa subunit. The optimal temperature and pH were 37°C and 7–8, respectively. The purified enzyme catalyzed hydrolysis of the acyl groups of both glycoglycerolipids and phospholipids, especially monogalacto-syldiacylglycerol and phosphatidylcholine. These results suggest that the enzyme hydrolyze the membrane lipids of the alga to release various saturated and unsaturated fatty acids, including arachidonic acid as substrate for prostaglandin synthesis.

The stereochemistry of glycerophospholipids (GPLs) has been of interest for its roles in the evolution of life and in their biological activity. However, because of their structural complexity, no convenient method to determine their configuration has been reported. In this work, through the first systematic application of vibrational circular dichroism (VCD) spectroscopy to various diacylated GPLs, we have revealed that their chirality can be assigned by the sign of a VCD exciton couplet generated by the interaction of two carbonyl groups. This paper also presents spectroscopic evidence for the stereochemistry of GPLs isolated from bacteria, eukaryotes, and mitochondria.

A simple and highly sensitive method for determining the fatty acid composition of food lipids containing conjugated linoleic acid (CLA) is described. The method is based on the separation of the 9-anthrylmethyl ester derivatives of saturated and unsaturated (conjugated and non-conjugated) fatty acids by reversed-phase high-performance liquid chromatography with fluorescence detection. Just like the other fatty acids, CLA reacts readily with 9-anthryldiazomethane at room temperature to produce 9-anthrylmethyl esters without isomerization and decomposition of the conjugated double bonds. Clear resolution of the individual fatty acids as their 9-anthrylmethyl esters is achieved on a highly efficient octadecylsilylated silica column (150- x 3-mm i.d., 3-microm particle size) using a stepwise gradient elution with methanol-water. The method is standardized with commercially available CLA isomers (cis-9, trans-11 and trans-10, cis-12-octadecadienoic acids, and their cis,cis and trans,trans isomers) and applied for determination of the fatty acid compositions of milk and sdairy products.

A simple and sensitive method for the determination of molecular species of bacterial phosphatidylglycerols (1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol, PG) as an intact form is described. The PG fraction isolated from Escherichia coli IAM12119 was subjected to reversed-phase high-performance liquid chromatography (HPLC) in conjunction with ion-trap mass spectrometry with electrospray ionization (ESI-MS). The HPLC analysis was performed with an ODS column (25 cm × 4.6 mm i.d., 5 μm particle size) using methanol/water/aqueous ammonium hydroxide (93 : 7 : 0.1, v/v/v) as the mobile phase, which gave a clear resolution of individual molecular species containing saturated, monounsaturated and cyclopropane fatty acids. In addition to a prominent deprotonated [M-H]− molecule, the negative ESI-MS gave weak product ions, [M-RCO]−, [M-H-RCOOH]− and [RCOO]−, by which individual molecular species could be identified. These product ions, derived from a fatty acid at the sn-2 position, showed greater intensities than those from the sn-1 position.

High-performance liquid chromatographic separation of enantiomeric 1,2- and 2,3-O-isopropylidene-sn-glycerols was performed on two different stationary phases of opposite configurations: N-(S)-1-(α-naphthyl)ethylaminocarbonyl-(S)-tert-leucine and N-(R)-1-(α-naphthyl)ethylaminocarbonyl-(R)-tert-leucine. This caused a reversal in the order of enantiomer elution. The isopropylideneglycerols were chromatographed as 3,5-dinitrophenylurethane derivatives. Complete enantiomer resolution, which permitted an accurate estimation of the optical purity of each enantiomer, was easily achieved on both columns (each 25 cm × 4.6 mm i.d.) within 15 min after an injection by an isocratic elution with hexane/dichloromethane/ethanol (40:12:3, by vol) as the mobile phase. The formations of diastereomeric π-π donor-accepter interaction, hydrogen bonding association, and dipole-dipole stacking between the solutes and the chiral stationary phases were considered as contributing factors to enantiomer resolution. Under optimal conditions, the optical purities of several commercially available 1,2-O-isopropylideneglycerol enantiomers were successfully determined in this study. The new method used in this study demonstrates that chiral phase high-performance liquid chromatography provides effective resolution, identification, and quantitation of synthetic 1,2- and 2,3-O-isopropylidene-sn-glycerol enantiomers.

An improved method for determining the composition of FFA in red tide flagellates by HPLC with fluorescence detection is described. For this purpose, total lipids from Heterosigma akashiwo, Chattonella antiqua, and C. marina were allowed to react with 9-anthryldiazomethane, then the resulting 9-anthrylmethyl esters of FFA were analyzed without any purification by RP-HPLC on a highly efficient C18 column (Superspher 100 RP-18e, 25 cm×4 mm i.d., 4 μm particle size; Merck, Darmstadt, Germany). Clear separations of long-chain saturated and unsaturated FFA, including 14∶0 and 16∶1, which were major components in the flagellates and were unresolved on a previously used C18 column, were achieved by a stepwise gradient elution using acetonitrile, water, and propan-2-ol. Two characteristic FA, 18∶5n−3 and trans-16∶1n−13, whose behaviors on RP-HPLC had not been reported previously, were also clearly separated from the other FFA. The FFA compositions of the flagellates determined by HPLC were in good agreement with those obtained by GLC. The present method is simple and sensitive, and would be widely applicable for compositional analysis of microalgal FFA.

In aquaculture centers of the northern region of Japan, "Nami-ita" (waved polycarbonate plates), on which the green alga Ulvella lens Crouan frat. (Chaetophoraceae: Chaetophorales) was cultured, are used to promote larval settlement and metamorphosis of the sea urchin species Strongylocentrotus intermedius (A. Agassiz) and S. nudus (A. Agassiz). We investigated chemical inducer(s) for larval settlement and metamorphosis of these sea urchins with extracts of U. lens. Bioassay-guided separation of the methanol extract using a combination of column and thin-layer chromatography led to the isolation of several active compounds, the chemical structures of which were determined by spectral and chemical methods. These active compounds were identified as glycoglycerolipids, all comprising several molecular species: sulfoquinovosyl monoacylglycerols (SQMGs), sulfoquinovosyl diacylglycerols (SQDGs), monogalactosyl monoacylglycerols (MGMGs), monogalactosyl diacylglycerols (MGDGs), digalactosyl monoacylglycerols (DGMGs) and digalactosyl diacylglycerols (DGDGs). Among these glycolipids, SQMGs, MGMGs, MGDGs and DGMGs induced larval metamorphosis of the sea urchin S. intermedius. SQMGs and MGDGs induced larval metamorphosis at a concentration of 5 µg ml–1, whereas SQDGs and DGDGs only induced larval settlement. These glycoglycerolipids are new congeners of chemical inducers to settlement and metamorphosis of planktonic larvae of sea urchins. The findings would provide a better understanding of larval settlement and metamorphosis in sea urchins.

Free fatty acid (FFA) level and galactolipase activity in a marine microalga causing red tides, Chattonella marina, were examined. The FFA content increased greatly during the growth from 2.8% of total lipids in the mid-logarithmic phase to 15.4% in the late stationary phase. A crude enzyme preparation derived from the alga had activities hydrolyzing the acyl groups of various glycoglycerolipids and phospholipids, especially monogalactosyldiacylglycerol (MGDG) molecules. The results suggest that FFAs in C. marina are released mainly from MGDG, which is a major lipid class in the alga, by galactolipase.

高速液体クロマトグラフィーと質量分析法を用いて, 生体膜の主要構成成分であるホスファチジルコリン (PC) の分子種の詳細を明らかにする高感度で簡便な方法を確立した. 植物油及び魚油から分離したPCを逆相HPLCとエレクトロスプレーイオン化質量分析法 (HPLC/ESI-MS) により分析した. HPLC分析では, 粒径4μmのODS充填剤を含むカラム (25cm×4mm i.d.) と少量の塩基を添加した移動相 {メタノール-アセトニトリル-トリエチルアミン (90 : 10 : 0.1, v/v/v)} を用いることによって, 高度不飽和酸を含む種々の分子種が明りょうに分離された. HPLC/ESI-MSでは, [M+H]+と[M+Na]+の強い分子量関連イオンと[M-RCO+2Na]+のフラグメントイオンが得られた. これらのイオンを利用して各分子種を同定した.

必須脂肪酸の一つであるγ-リノレン酸のトリアシルグリセロール(TG)分子中での結合位置を決定する簡便な方法を検討した。糸状菌Mucor circinelloides及びその変異株の脂質からγ-リノレン酸高含有TGを分離し,これをグリニャール分解してsn-1,2-及びsn-2,3-ジアシルグリセロール(DG)のエナンチオマー混合物を得た。この混合物を3,5-ジニトロフェニルウレタン誘導体に変換した後,(R)-1-(1-ナフチル)エチルアミンを不斉部位とするキラル固定相を装備したHPLCを用いて光学分割し,純粋なsn-1,2-DG(I)及びsn-2,3-DG(II)画分を得た。IとII及びTGの脂肪酸組成からTG分子中のsn-1,sn-2及びsn-3位におけるγ-リノレン酸の分布を算出した。また,IとIIを逆相HPLCで分析して,それぞれの分子種組成を明らかにした。得られた結果から計算により求めたTGの脂肪酸組成はオリジナルTGのそれと近似したことから,本法の正確さを良好であると判断された。Mucor circinelloidesの産生するγ-リノレン酸はTG分子中のsn-3位に最も多く存在し,次いでsn-1位,そしてsn-2位であり,大きく偏って存在することが明らかとなった。