Eicosapentaenoic acid and arachidonic acid: collaboration and not antagonism is the key to biological understanding.

Much of the literature on omega-3 and omega-6 fatty acids suggests that desirable effects of omega-3 fatty acids are in part related to depletion of arachidonic acid (AA). However, in rats and humans, we have found that low doses of EPA actually elevate membrane AA phospholipid concentrations. In patients with schizophrenia, treatment with eicosapentaenoic acid (EPA) produced clinical improvement, but that improvement was greater at a dose of 2 g/day than at 4 g/day. The improvement was not significantly correlated with changes in either EPA or docosahexaenoic acid (DHA) but was highly significantly positively correlated with rises in red cell membrane AA. We suggest that elevation of concentrations of both AA and EPA in cell membranes may be important for health.

Mechanisms for ion formation during the electron impact-mass spectrometry of picolinyl ester and 4,4-dimethyloxazoline derivatives of fatty acids.

Mass spectral studies have been conducted with isotopically stable labelled and fluorinated picolinyl esters and 4,4-dimethyloxazoline (DMOX) derivatives of fatty acids in order to establish mechanisms of ion formation. Reciprocal hydrogen transfer is shown to be involved in the formation of the ion at m/z 126 with dimethyloxazoline derivatives and for the ion at m/z 164 with picolinyl esters. Inclusion of a fluorine atom alpha to the carboxyl of a fatty acid has been demonstrated to enhance rearrangements for expulsion of internal chain fragments with both methyl ester and dimethyloxazoline derivatives. When two fluorine atoms are inserted into the alpha position a similar rearrangement has been shown to occur with picolinyl esters, although not nearly to the same extent as that observed with either of the other derivatives. Mechanisms for such rearrangements are proposed and discussed. With fatty acid dimethyloxazoline derivatives the M-15 ion arises solely from the loss of a methyl radical from the ring and the M-43 ion has at least three different mechanisms of formation. Such rearrangements make it difficult to establish the identity of the terminal moiety of the alkyl chain. In mass spectrometry terms the picolinyl ester would seem to be the superior derivative for structural characterisation of fatty acids.

The effect of dietary trans alpha-linolenic acid on plasma lipids and platelet fatty acid composition: the TransLinE study.

OBJECTIVE: To collect (i) baseline data and (ii) execute a large multicentre study examining the effect of trans alpha-linolenic acid on its incorporation into plasma lipids and on risk factors for coronary heart disease. DESIGN: Male volunteers were recruited and the habitual diet assessed by a 4-d weighed record. Fatty acid composition of plasma and platelet lipids were determined by gas chromatography at baseline. After a 6 week run-in period on a trans 'free' diet, male volunteers were randomised to consume 0.6 % of energy trans alpha-linolenic acid or to continue with a diet 'low' in trans alpha-linolenic acid for 6 weeks. SETTING: Three European university research departments supported by the research and development departments of the food industry. SUBJECTS: Male volunteers (88) recruited by local advertisement. METHODS: Replacement of 30 % of the fat of the habitual diet by margarine, oil and foods. Rapeseed oil was deodorised especially to produce the trans 'free' and 'high' trans foods for this study. The incorporation and conversion of trans alpha-linolenic acid into plasma lipids and platelets was assessed by gas chromatography and dietary compliance was verified by 4-d weighed record. RESULTS: Less trans alpha-linolenic acid isomers are incorporated into human plasma lipids in French volunteers than in Dutch or Scottish volunteers consuming their habitual diets. Trans 'free' alpha-linolenic acid-rich oil can be produced by careful deodorization during refining. The 'high' trans diet provided 1410+/-42 mg/d trans isomers of alpha-linolenic acid, whilst the 'low' trans group consumed 60+/-75 mg/d. The change in plasma lipid and platelet fatty acid composition documented that trans linolenic isomers are incorporated and converted to a trans isomer of eicosapentaenoic acid. Only the 15-trans alpha-linolenic acid is incorporated into plasma cholesteryl esters. The group consuming low trans diet had a slightly higher intake of fat, especially saturated and monounsaturated fat. CONCLUSIONS: Trans 'free' rapeseed oil, rich in alpha-linolenic acid, can be produced by careful deodorization. Dietary records show good compliance. Dietary trans isomers of alpha-linolenic acid are incorporated in plasma lipids and converted to long-chain polyunsaturated fatty acids. Their effects on risk factors for coronary heart disease and their metabolism will be reported elsewhere. SPONSORSHIP: European Commission (FAIR 95-0594 grant). European Journal of Clinical Nutrition (2000) 54, 104-113

Arachidonic, eicosapentaenoic, and biosynthetically related fatty acids in the seed lipids from a primitive gymnosperm, Agathis robusta.

The fatty acid composition of the seeds from Agathis robusta, an Australian gymnosperm (Araucariaceae), was determined by a combination of chromatographic and spectrometric techniques. These enabled the identification of small amounts of arachidonic (5,8,11,14-20:4) and eicosapentaenoic (5,8,11,14,17-20:5) acids for the first time in the seed oil of a higher plant. They were apparently derived from gamma-linolenic (6,9,12-18:3) and stearidonic (6,9,12,15-18:4) acids, which were also present, via chain elongation and desaturation, together with other expected biosynthetic intermediates [bis-homo-gamma-linolenic (8,11,14-20:3) and bishomo-stearidonic (8,11,14,17-20:4) acids]. Also present were a number of C20 fatty acids, known to occur in most gymnosperm families, i.e., 5,11-20:2, 11,14-20:2 (bishomo-linoleic), 5,11,14-20:3 (sciadonic), 11,14,17-20:3 (bishomo-alpha-linolenic), and 5,11,14,17-20:4 (juniperonic) acids. In contrast to most other gymnosperm seed lipids analyzed so far, A. robusta seed lipids did not contain C18 delta5-desaturated acids [i.e., 5,9-18:2 (taxoleic), 5,9,12-18:3 (pinolenic), or 5,9,12,15-18:4 (coniferonic)]. These structures support the simultaneous existence of delta6- and delta5-desaturase activities in A. robusta seeds. The delta6-ethylenic bond is apparently introduced into C18 polyunsaturated acids, whereas the delta5-ethylenic bond is introduced into C20 polyunsaturated acids. A general metabolic pathway for the biosynthesis of unsaturated fatty acids in gymnosperm seeds is proposed. When compared to Bryophytes, Pteridophytes (known to contain arachidonic and eicosapentaenoic acids), and species from other gymnosperm families (without such acids), A. robusta appears as an "intermediate," with the C18 delta6-desaturase/C18-->C20 elongase/C20 delta5-desaturase system in common with the former subphyla, and the unsaturated C18-->C20 elongase/C20 delta5-desaturase system specific to gymnosperms. The following hypothetical evolutionary sequence for the C18 delta6/delta5-desaturase class in gymnosperm seeds is suggested: delta6 (initial)-->delta6/delta5 (intermediate)-->delta5 (final).

Delta5-olefinic acids in the seed lipids from four Ephedra species and their distribution between the alpha and beta positions of triacylglycerols. Characteristics common to coniferophytes and cycadophytes.

The fatty acid compositions of the seed lipids from four Ephedra species, E. nevadensis, E. viridis, E. przewalskii, and E. gerardiana (four gymnosperm species belonging to the Cycadophytes), have been established with an emphasis on delta5-unsaturated polymethylene-interrupted fatty acids (delta5-UPIFA). Mass spectrometry of the picolinyl ester derivatives allowed characterization of 5,9- and 5,11-18:2; 5,9,12-18:3; 5,9,12,15-18:4; 5,11-20:2; 5,11,14-20:3; and 5,11,14,17-20:4 acids. Delta5-UPIFA with a delta11-ethylenic bond (mostly C20 acids) were in higher proportions than delta5-UPIFA with a delta9 double bond (exclusively C18 acids) in all species. The total delta5-UPIFA content was 17-31% of the total fatty acids, with 5,11,14-20:3 and 5,11,14,17-20:4 acids being the principal delta5-UPIFA isomers. The relatively high level of cis-vaccenic (11-18:1) acid found in Ephedra spp. seeds, the presence of its delta5-desaturation product, 5,11-18:2 acid (proposed trivial name: ephedrenic acid), and of its elongation product, 13-20:1 acid, were previously shown to occur in a single other species, Ginkgo biloba, among the approximately 170 gymnosperm species analyzed so far. Consequently, Ephedraceae and Coniferophytes (including Ginkgoatae), which have evolved separately since the Devonian period (approximately 300 million yr ago), have kept in common the ability to synthesize C18 and C20 delta5-UPIFA. We postulate the existence of two delta5-desaturases in gymnosperm seeds, one possibly specific for unsaturated acids with a delta9-ethylenic bond, and the other possibly specific for unsaturated acids with a delta11-ethylenic bond. Alternatively, the delta5-desaturases might be specific for the chain length with C18 unsaturated acids on the one hand and C20 unsaturated acids on the other hand. The resulting hypothetical pathways for the biosynthesis of delta5-UPIFA in gymnosperm seeds are only distinguished by the position of 11-18:1 acid. Moreover, 13C nuclear magnetic resonance spectroscopy of the seed oil from two Ephedra species has shown that delta5-UPIFA are essentially excluded from the internal position of triacylglycerols, a characteristic common to all of the Coniferophytes analyzed so far (more than 30 species), with the possibility of an exclusive esterification at the sn-3 position. This structural feature would also date back to the Devonian period, but might have been lost in those rare angiosperm species containing delta5-UPIFA.

Tanacetum (Chrysanthemum) corymbosum seed oil--a rich source of a novel conjugated acetylenic acid.

A new conjugated trans,trans-diunsaturated acetylenic acid (17%), found for the first time in nature as a main component of the seed oil of Tanacetum (Chrysanthemum) corymbosum, was shown to be octadeca-8t,10t-dien-12-ynoic acid. Another already known naturally occurring acetylenic acid, crepenynic acid (10%), was found also in the seed oil. The structure of this new unusual fatty acid was confirmed by chromatographic (thin-layer chromatography, gas chromatography, high-performance liquid chromatography) and spectroscopic (infrared, ultraviolet, mass spectrometry) methods by using different chemical derivatizations (deuteration, preparation of picolinyl ester, dimethyloxazoline, and maleic anhydride adduct).

Gas chromatography-mass spectrometry methods for structural analysis of fatty acids.

Procedures for structural analysis of fatty acids are reviewed. The emphasis is on methods that involve gas chromatography-mass spectrometry and, in particular, the use of picolinyl ester and dimethyloxazoline derivatives. These should be considered as complementing each other, not simply as alternatives. However, additional derivatization procedures can be of value, including hydrogenation and deuteration, and preparation of dimethyl disulfide and 4-methyl-1,2,4-triazoline-3,5-dione adducts. Sometimes complex mixtures must be separated into simpler fractions prior to analysis by gas chromatography-mass spectrometry. Silver ion and reversed-phase high-performance liquid chromatography are then of special value. In particular, a novel application of the latter technique, involving a base-deactivated stationary phase and acetonitrile as mobile phase, is described that is suited to the separation of fatty acids in the form of picolinyl ester and dimethyloxazoline derivatives, as well as methyl esters.

Liquid chromatography with particle-beam electron-impact mass spectrometry of diacylglycerol nicotinates.

Several synthetic diacylglycerols and natural mixtures derived from soybean, egg yolk, and bovine liver phosphatidylcholines were examined in the form of nicotinate derivatives by liquid chromatography with particle-beam electron-impact mass spectrometry. High-performance liquid chromatography was carried out in the reversed-phase mode with a base-deactivated octyl-/octadecylsilyl stationary phase. The nature (size and degree of unsaturation) of the acyl residues was readily determined from the mass spectra. Uniquely, the positions of the double bonds could be deduced, although this became increasingly difficult as the degree of unsaturation of diacylglycerols containing mixed acyl residues increased. Reverse 1,2-diacylglycerol isomers could be distinguished by their mass spectra.

The unusual occurrence of 14-methylhexadecanoic acid in Pinaceae seed oils among plants.

14-Methylhexadecanoic (14-MHD) acid has been identified in a sample of pine seed oil (Pinus contorta) by gas-liquid chromatography-mass spectrometry of its picolinyl ester derivative. Its identification (through its equivalent chain length) and its distribution in four conifer families have been checked. It occurred only in Pinaceae, where it was found in 72 species belonging to the genera Pinus, Abies, Cedrus, Tsuga, Pseudotsuga, Larix, and Picea, in the range 0.02-1.15%. 14-MHD acid could not be detected in the lipids of Taxaceae (Taxus baccata), Cupressaceae (Juniperus communis), or Taxodiaceae (Sciadopytis verticillata), even after a 10-fold concentration of the saturated acid fraction isolated by argentation thin-layer chromatography. It is concluded that Pinaceae, along with Ginkgo biloba seed lipids, are major exceptions in the plant kingdom with regard to 14-MHD acid, which otherwise occurs almost exclusively in lipids of animals and microorganisms. The biosynthesis and metabolic role of 14-MHD acid, which otherwise also occur in wood and leaf lipids, remain unknown.

Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of delta6-desaturated fatty acids in transgenic tobacco.

gamma-Linolenic acid (GLA; C18:3 delta(6,9,12)) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a delta6-fatty acid desaturase using linoleic acid (C18:2 delta(9,12)) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the delta6-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 delta(6,9,12,15)) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage delta6-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b5.

Metabolites of conjugated isomers of linoleic acid (CLA) in the rat.

Metabolism of conjugated isomers of linoleic acid (CLA) in rats was studied by feeding high quantities of CLA (180 mg/day) for six days to animals that had been reared on a fat-free diet for two weeks. After this period, animals were sacrificed and liver lipids extracted. High-performance liquid chromatography (HPLC) analyses with UV detection revealed the presence of conjugated polyunsaturated fatty acids in the total liver lipid methyl esters. After isolation by HPLC, three fatty acid metabolites were identified by gas liquid chromatography coupled with mass spectrometry as being C20:3 delta 8,12,14, C20:4 delta 5,8,12,14 and C20:4 delta 5,8,11,13. A higher quantity of C20:4 delta 5,8,12,14 compared to C20:4 delta 5,8,11,13 was observed. These must arise from the elongation and desaturation of 18:2 delta 10,12 and 18:2 delta 9,11, respectively.

Novel di-, tri-, and tetraenoic fatty acids with bis-methylene-interrupted double-bond systems from the sponge Haliclona cinerea.

Unusual fatty acids, with up to 34 carbon atoms and containing one or two bis-methylene-interrupted double-bond systems, have been identified in the sponge Haliclona cinerea from the Black Sea. These include the dienes-5,9-16:2, 7,11-18:2, 9,13-20:2, 13,17-24:2, 15,19-26:2, 17,21-28:2, 19,23-30:2 and 21,25-32:2; trienes-5,9,23-30:3, 5,9,24-31:3, 5,9,25-32:3, and 5,9,27-34:3; and the tetraenes-5,9,19,23-30:4, 5,9,21,25-32:4, and 5,9,23,27-34:4. In addition, 5,9,13-eicosatrienoic acid was present. Many of these do not appear to have been described before, and only 5,9-16:2 and 5,9,23-30:3 are found often in sponges. They were identified by using silver-ion high-performance liquid chromatography to simplify the complex mixture of fatty acids for subsequent analysis by gas chromatography-mass spectrometry as picolinyl ester derivatives. Deuteration with Wilkinson's catalyst in homogeneous solution confirmed the structures. We speculate that the di- and tetraenoic fatty acids arise by chain elongation of 5,9-hexadecadienoic acid, also a major component of the lipids, followed by further insertion of double bonds in the 5 and 9 positions. The trienes may be formed from 9-hexadecenoic acid by similar mechanisms.

Silver ion chromatography of lipids and fatty acids.

Silver ion chromatography as applied to the analysis of lipids is reviewed. Thin-layer, column, high-performance liquid and supercritical fluid chromatography in the silver ion mode are included. The lipid types covered are fatty acids, triacylglycerols and complex lipids. Separations are divided into those according to number, geometry and position of double bonds, as well as acyl positional isomers for triacylglycerols. The mechanism of silver ion chromatography is discussed in relation to recent studies using silver ion high-performance liquid chromatographic methodology.

Expression of mRNA and steady-state levels of protein isoforms of enoyl-ACP reductase from Brassica napus.

The expression of mRNA and the steady-state levels of two-component enzymes of plant fatty acid synthetase (FAS) were studied. Northern analysis of enoyl-ACP reductase (ER) and stearoyl-ACP desaturase (SD) gene expression showed that steady-state levels of both transcripts increase during lipid deposition in the seed reaching a maximum at 29 days after flowering (DAF). The steady-state level of ER message falls very quickly after reaching its maximum, whereas the SD message is longer-lived. The levels of these specific mRNAs in seed are 15-30 times greater than in leaf. Optimum mRNA expression precedes the maximum levels of synthesis of the two proteins, which in turn precede the maximum level of oil. The expression of isoenzymes of ER were examined by two-dimensional western blotting in both leaf and seed tissue. Four enzymes are expressed in both of these tissues; the two most abundant isoforms in seed material are also the most abundant in leaf tissue.

An unusual fatty acid composition for a fresh-water mussel, Unio tumidus, from Bulgaria.

A combination of hplc in the silver ion mode and gc-ms of picolinyl ester derivatives was used to identify the fatty acids in a fresh-water mussel, Unio tumidus, from the Danube in Bulgaria. A number of novel fatty acids were found, including 14-methylpentadec-6-enoic and 17-methyloctadec-8-enoic acids. Eicos-7-enoic acid was a major component.

Extraction of plasma triacylglycerols by the mammary gland of the lactating cow.

The fatty acid content of triacylglycerols (TG) in arterial and mammary venous blood plasma has been analysed in five cows that were close to peak lactation. The fatty acids in arterial TG were extracted by the mammary gland in amounts 14:0 less than 16:0 less than 18:0 and 18:0 greater than 18:1n - 9 greater than 18:2. The difference of extraction between palmitate and stearate was similar in different TG species. The fatty acid 18:1n - 7, which was largely the trans isomer, was extracted more than 18:1n - 9. Significant amounts of phytanic acid were acylated in plasma TG, but not extracted by the mammary gland.

Separation of cholesterol esters by silver ion chromatography using high-performance liquid chromatography or solid-phase extraction columns packed with a bonded sulphonic acid phase.

Two methods for the separation of cholesterol esters, based on the number of double bonds in their fatty acid moieties, are presented. Silver ion chromatography, usually performed on thin-layer chromatographic plates, was made suitable for high-performance liquid chromatography (HPLC) and solid-phase extraction. Separation on a bonded sulphonic acid phase loaded with silver ions was achieved with cholesterol esters containing up to six double bonds in their fatty acid moieties. No cross-contamination between fractions with different numbers of double bonds was detected with the HPLC method, was demonstrated by subsequent gas chromatographic analysis of the fatty acid moieties, following transmethylation. For adequate separations with the solid-phase extraction columns it proved important to avoid overloading. The methods may be of use for the off-line analyses of the sterol compositions of the isolated fractions, which each contain sterol esters with an equal number of double bonds in their fatty acid moieties.