CTA ester bonds in TG molecules were not attacked by pancreatic lipase and lipases produced by microbes such as Candida cylindracea, Chromobacterium viscosum, Geotricum candidium, Pseudomonas fluorescens, Rhizophus delemar, R. arrhizus and Mucor miehei. An aliquot of total TG of all the seed oils and each TG fraction of the oils collected from HPLC runs were deuterated prior to partial hydrolysis with Grignard reagent, because CTA molecule was destroyed with treatment of Grignard reagent. Deuterated TG (dTG) was hydrolyzed partially to a mixture of deuterated diacylglycerols (dDG), which were subsequently reacted with (S)-(+)-1-(1-naphthyl)ethyl isocyanate to derivatize into dDG-NEUs. Purified dDG-NEUs were resolved into 1, 3-, 1, 2- and 2, 3-dDG-NEU on silica columns in tandem of HPLC using a solvent of 0.4% propan-1-o1 (containing 2% water)-hexane. An aliquot of each dDG-NEU fraction was hydrolyzed and (fatty acid-PFB ester). These derivatives showed a diagnostic carboxylate ion, (M-1)-, as parent peak and a minor peak at m/z 196 (PFB-CH3)- on NICI mass spectra. In the mass spectra of the fatty acid-PFB esters of dTGs derived from the seed oils of T. kilirowii and M. charantia, peaks at m/z 285, 287, 289 and 317 were observed, which corresponded to (M-1)- of deuterized oleic acid (d2-C18:0), linoleic acid (d4-C18:0), punicic acid (d6-C18:0) and eicosamonoenoic acid (d2-C20:0), respectively. Fatty acid compositions of deuterized total TG of each oil measured by relative intensities of (M-1)- ion peaks were similar with those of intact TG of the oils by GLC. The composition of fatty acid-PFB esters of total dTG derived from the seed oils of T. kilirowii are as follows; C16:0, 4.6 mole % (4.8 mole %, intact TG by GLC), C18:0, 3.0 mole % (3.1 mole %), d2C18:0, 11.9 mole % (12.5 mole %, sum of C18:1Ω9 and C18:1Ω7), d4-C18:0, 39.3 mole % (38.9 mole %, sum of C18:2Ω6 and its isomer), d6-C18:0, 41.1 mole % (40.5 mole %, sum of C18:3 9c,11t,13c, C18:3 9c,11t,13r and C18:3 9t,11t,13c), d2-C20:0, 0.1 mole % (0.2 mole % of C20:1Ω9). In total dTG derived from the seed oils of M. charantia, the fatty acid components are C16:0, 1.5 mole % (1.8 mole %, intact TG by GLC), C18:0, 12.0 mole % (12.3 mole %), d2-C18:0, 16.9 mole % (17.4 mole %, sum of C18:1Ω9), d4-C18:0, 11.0 mole % (10.6 mole %, sum of C18:2Ω6), d6-C18:0, 58.6 mole % (57.5 mole %, sum of C18:3 9c,11t,13t and C18:3 9c,11t,13c). In the case of Aleurites fordii, C16:0; 2.2 mole % (2.4 mole %, intact TG by GLC), C18:0; 1.7 mole % (1.7 mole %), d2-C18:0; 5.5 mole % (5.4 mole %, sum of C18:1Ω9), d4-C18:0 ; 8.3 mole % (8.5 mole %, sum of C18:2Ω6), d6-C18:0; 82.0 mole % (81.2 mole %, sum of C18:3 9c,11t,13t and C18:3 9c,11t,13c). In the stereospecific analysis of fatty acid distribution in the TG species of the seed oils of T. kilirowii, C18:3 9c,11t,13r and C18:2Ω6 were mainly located at sn-2 and sn-3 position, while saturated acids were usually present at sn-1 position. And the major molecular species of (C18:2Ω6)(C18:3 9c,11t,13c)2 and (C18:1Ω9)(C18:2Ω6)(C18:3 9c,11t,13c) were predominantly composed of the stereoisomer of sn-1-C18:2Ω6, <..

In search for several fatty acid with unusual structure in vegetable oils, we have found that unknown peaks were shown on GLC in the analysis of fatty acids of the lipids from the pulp of ripened jujube (Zizypus jujuba var. inermis) fruits. These fatty acids were identified as a series of cis-monoenoic acids with Ω-5 double bond system such as C14:1Ω5, C16:1Ω5 and C18:1Ω5, including Ω-7 fatty acid as C16:1Ω7 and C18:1Ω7, by GLC, solid-phase extraction silver ion-column chromatographic, GLC-mass spectrometric and IR techniques. First of all, total fatty acid methyl esters were resolved into saturated and branched fatty acid, monoenoic acid, dienoic acid, and trienoic acid fraction, respectively, with 100% dichloromethane (DCM), DCM/acetone (9:1, v/v) 100% acetone, and acetone/ acetonitrile (97:3, v/v) solvent system. Unknown fatty acids were included in the monoenoic fraction and were confirmed to have cis-configuration by IR. Picolinyl esters of monoenoic fatty acids gave distinct molecular ion peak and dominant diagnostic peaks, for example, m/z 317, 220 and 260 fragment for cis-C14:1Ω5, m/z 345, m/z 248 and 288 fragment for cis-C16:1Ω5 and m/z 373, m/z 276 and 316 fragment for cis-C18:1Ω5. In this way the occurrence of cis-C16:1Ω7 and cis-C18:1Ω7 could be deduced from the appearance of prominent fragments as m/z 345, 220 and 260, and m/z 373, 248 and 280. Level of total Ω-5 fatty acids amounted to about 30% in the fatty acid composition with the predominance of C16:1Ω5 (18.7~25.0%), in the semi-ripened and/or ripened samples collected in September 14 (C16:1Ω5 ; 18.7%, C14:1Ω5 ; 3.6% and C18:1Ω5 ; 3.0%), September 22 (C16:1Ω5 ; 25.0%, C14:1Ω5 ; 1.4% and C18:1Ω5 ; 2.6%), and October 7 (C16:1Ω5 ; 24.7%, C14:1Ω5 ; 7.7% and C18:1Ω5 ; 2.5%). However, the lipids extracted from unripened jujube in July and August contain these unusual fatty acids as low as negligible. It could be observed that the level of Ω-5 fatty acids in the pulps increased sharply with an elapse of ripening time of jujube fruits. Other monoenoic fatty acids with Ω-7 series, C16:1Ω7 (palmitoleic acid) and C18:1Ω7 (cis-vaccenic acid) could be detected. And in the lipids of the kernel and leaf of jujube, none of Ω-5 fatty acids could be detected.

We checked the presence of phospholipase A2(PLA)2 which could split the ester bond at the position 2 in the glycerol backbone of glycerophospholipids, in the cells of hyperthermophiles of Pyrococcus horikoshii and Sulfolobus acidocaldarius. The results obtained are as follows; (1). Pyrococcus horikoshii cells were grown in obligate anaerobic conditions at 95℃ and they needed sulfur as energy source instead of oxygen, while Sulfolobus acidocaldarius species grew well in the aerobic medium (pH 2.5) containing yeast and sucrose at 75℃. (2). Pyrococcus horikoshii cells produced phospholipase A2 in the cell culture media although this species did not show lipase activity at least in the pH range of 1.5 ~ 3.5. Sulfolobus acidocaldarius cells produced lipase hydrolyzing triacylglycerols such as triolein, but did not split any kind of phospholipids used as substates. (3). The compound of 1-decanoyl-2-(p-nitrophenylglutaryl) phosphatidylcholine was not suitable for a substrate in this experiment, though frequently used as a subtrate for checking presence of phospholipase A2, for its decomposi-tion in this experiment. The L-α-phosphatidylcholine-β-[N-7-nitrobenz-2-oxa-1, 3-diazol]aminohexanoyl-γ-hexadecanoyl labelled with a fluorescent material, did not show any migration of acyl chains in the molecule during the reaction with phospholipase A2 under a hot condition. (4). Phospholipase A2 in the cells of Pyrococcus horikoshii, showed the optimum activity at pH6.7~7.2 and 95~105℃, respectively, and was activated by addition of calcium chloride solution. Andthe phospholipase A2 specifically hydrolyzed glycero-phospholipids such as phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and phosphatidyl inositol, but could not split phospholipid containing ether bonds in the molecule such as DL -α-phosphatidylcholine-β-palmitoyl-γ-O-hexadecyl, DL-α-phosphati- dylcholine-β- oleoyl-γ-O-hexadecyl, DL-phosphatidylcholine-dihexadecyl.

All the triacylglycerols including the molecular species having δ5-unsaturated fatty acids from the seeds of Pinus Koraiensis, were split into a mixture of diacylglycerols by a Grignard reagent prepared with allyl bromide without arousing acyl chains of a glycerol moiety to migration, and were also easily partially hydrolyzed to diacylglycerols by pancreatic lipase. (S)-(+)-(1-naphthyl)ethyl urethane(NEU) derivatives of the diacylglycerol mixture derived from the triacylglycerols were fractionated into sn-1, 3-, sn-1, 2- and sn-2, 3-DG-NEU by silica-HPLC and the fatty acid composition of these fractions was analysed. C18:1Ω9 is distributed evenly in the three positions of TG with C18:2Ω6 mainly located in sn-2 position, while δ5-unsaturated fatty acids such as δ5.9-C18:2, δ5.9.12-C18:3 and δ5.11.14-C20:3 are exclusively present in the sn-3 position. These results could be confirmed by 13C-NMR spectroscopy : the signals at δ173.231 ppm and δ172.811 ppm of the carbonyl carbon of acyl moieties indicate the presence of saturated acids and/or C18:1Ω9 (oleic acid) in the α(α')- or β- positions, and C18:2Ω6 including C18:1Ω9 in the β-position, respectively. In addition, the resonance at δ173.044 ppm suggested a location of δ5-unsaturated fatty acid moiety in the α(α')-position.

The lipids from the seeds of Pinus koraiensis mostly composed of triacylglycerols (TGs), in which linoleic acid (46.2 mol%) and oleic acid (25.6 mol%) are present as main components in the fatty acid composition. Surprisingly, they also have unusual fatty acids with δ5-double bond systems such as δ5.9.12-C18:3 (16.0 mol%), δ5.9-C18:2 (2.3 mol%) and δ5.11.14-C20:3 (0.8 mol%). Saturated fatty acids of palmitic, stearic and arachidic acid were present in less than 8.0 mol%. TG was resolved into 17 fractions by reverse-phase HPLC according to so-called partition number (PN) suggested by Plattner, in which TG molecules with δ5-NMDB acyl chains eluted later than did those with δ9-MDB acyl radicals. Ag+-HPLC separated the TG into 14 fractions more clearly than did those with δ9-MDB acyl radicals. Ag+-HPLC separated the TG into 14 fractions more clearly than did reverse-phase HPLC, and the complexity of δ5.9.12-C18:3 moiety with silver ion impregnated in the column bed was in the level between δ9.12.15-C18:3 (C18:3Ω3) and C18:2Ω6 (δ9.12-C18:2). In the Ag+-HPLC, it was found that the molecular species having a given-numbered double bonds widely spreaded in the acyl chains eluted earlier than those concentrated in one acyl chain. The main molecular species are (C18:2Ω6)2/δ5.9.12-C18:3 (14.8 mol%), C18:1Ω9/C18:2Ω6)2 (12.8 mol%) and C18:1Ω9/C18:2Ω6/δ5.9.12-C18:3 (10.9 mol%).

Triacylglycerols of the seeds of Ginkgo biloba have been resolved by high-performace liquid chromatography(HPLC in the silver-ion and reverse-phase modes. The fatty acids were identified by a combination of capillary gas chromatography and gas-chromatography /mass spectrometry as the methyl and /or picolinyl ester. The main components are C18:2Ω6(39.0mol%), C18:1Ω7(asclepic acid 21.5mol%), and C18:1Ω9(oleic acid, 13.8mol%). Considerable amounts of unusual acid such as C20:3δ5,11,14 (5.7mol%), C18:2δ5,9(2.8mol%), and C18:3δ5,9,12(1.6mol%), were checked. In addition, an anteiso-branched fatty acid, 14-methylhexadecanoic acid, was also present as a minor component(0.9 mol%). The triacylglycerols were separated into 17 fractions by reverse-phase HPLC, and the fractionation was achieved according to the partition numnber(PN) in which a δ5-non methylene interrupted double bond(5-NMDB) showed different behaviour from a methylene interrupted double bond in a molecule with a given cahinlength. Silver-ion HPLC exhibited excellent resolution in which fractions(23 fractions) were resolved on the basis of the number and configuration of double bonds. In this instance, the strength of interaction of a δ5-NMDB system with silver ions seemed to be weaker than a methylene interrupted double bond system. The principal triacylglycerol species are as follows ; (C18:2Ω6)2/C18:1Ω7, C18:1Ω9/C18:1Ω7/C18:2Ω6, (C18:1Ω7)2/C18:2Ω6, C16:1Ω7/C18:1Ω9/C20:3δ5,11,14, C16:1Ω7/C18:1Ω7/C20:3δ5,11,14, C18:1Ω9/C18:1Ω7/C18:2Ω6, C18:1Ω9/C18:2δ5,5/C20:3δ5,11,14, (C18:1Ω7)2/C18:2Ω6 and (C18:1Ω9)2/C18:2Ω6, while simple triacylglycerols without C18:2Ω6)3 were not present. Stereospecific analysis showed that fatty acids with δ5-NMDB system and saturated chains were predominantly located at the site of sn-3 carbon of glycerol backbones. It is evident that there is asymmetry in the distribution of fatty acids in the TG molecules of Ginkgo nut oils.