Gary Dobson

Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants.

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the ω3/6 Δ8-desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Δ9-specific elongating activity from Isochrysis galbana, a Δ8-desaturase from Euglena gracilis and a Δ5-desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Δ9-elongating activity, which may bypass rate-limiting steps present in the conventional Δ6-desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.

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.

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.

Isomers in commercial samples of conjugated linoleic acid

In connection with our analytical activities (MRS Lipid Analysis Unit), we have analyzed several commercial samples of conjugated linoleic acid (CLA). Most of these have been prepared by alkali-isomerization of linoleic acid or of oils, such as sunflower or safflower, rich in this acid. (i) We have examined the methyl esters, which must be prepared from the acids avoiding the use of acidic catalysts, by gas chromatography (GC). With a 25-m carbowax capillary column, most of the samples show two major peaks which are well-resolved from each other, along with minor peaks running later which are first the all-cis and then the all-trans dienes. The two major peaks are usually considered to be only the 9c,11t and 10t,12c isomers. This interpretation is not consistent with the GC/mass spectrometry (MS) data reported below. When examined on a highly polar 100-m capillary column (CP Sil 88), the GC trace is more complex. Several samples show a new peak for 11,13 diene, and some indicate the presence of several other isomers. We also have evidence that the 9,11 peak contains the 8,10 isomer though we have been unable to resolve these. (ii) GC–MS of the diene adducts formed through reaction with 4-methyl2,3,4-triazoline-3,5-dione (MTAD derivatives), with selected ion monitoring, shows the presence of 8,10; 9,11; 10,12; and 11,13 dienes (1). For example, Figure 1 illustrates the selected ion chromatogram for one of the diagnostic ions from each isomer in a commercial CLA preparation. It is clear that four unresolved isomers are present. The proportions of these vary widely (presumably depending on the conditions of alkali-isomerization) and even more isomers are sometimes present. GC–MS of the dimethyloxazoline derivatives confirmed the identity of the major products. (iii) High-resolution 13C nuclear magnetic resonance spectroscopy confirmed the presence of at least four cis,trans conjugated dienes and gave quantitative results in line with those obtained by GC–MS analysis. We conclude that most samples of CLA, though rich in the 9,11 (probably mainly if not entirely the 9c,11t isomer) and 10,12 dienes (probably mainly if not entirely the 10t,12c isomer) also contain at least the 8,10 and 11,13 cis,trans dienes, sometimes at quite high levels. These are accompanied by allcis and all-trans dienes. In one commercial sample of CLA, we found the following dienes: 8,10 (14%), 9,11 (30%), 10,12 (31%), and 11,13 (24%). It is important that those who produce these materials and use them for research purposes appreciate the complex nature of their products. To our knowledge, the identity of the biologically active CLA is not known although it is generally assumed to be 9c,11t-18:2. Nor is it known how the activity of this isomer may be influenced by the presence of other isomeric CLA.

A practical guide to the isolation, analysis and identification of conjugated linoleic acid.

Natural and synthetic conjugated linoleic acids (CLA) are reputed to have therapeutic properties that are specific to particular geometrical and positional isomers. Analysis of these has presented unique problems that have brought forward distinctive solutions, especially the use of gas chromatography–mass spectrometry and silver-ion high-performance liquid chromatography. In the analysis of CLA present at low levels in tissue samples, it is sometimes necessary to use concentration methods. In this review, the most useful and practical methods for the isolation and analysis of CLA isomers in tissues and in commercial CLA preparations are described.

Anthocyanin-enriched bilberry and blackcurrant extracts modulate amyloid precursor protein processing and alleviate behavioral abnormalities in the APP/PS1 mouse model of Alzheimer's disease

A growing body of epidemiological evidence suggests that fruit and vegetable juices containing various phenolic compounds can reduce the risk of Alzheimers disease (AD). As the altered amyloid precursor protein (APP) processing leading to increased β-amyloid (Aβ) production is a key pathogenic feature of AD, we elucidated the effects of different polyphenols on neuroprotection and APP processing under different in vitro stress conditions. The effects of these compounds were also investigated in transgenic AD mice (APdE9). Free radical toxicity and apoptosis were induced in human SH-SY5Y neuroblastoma cells overexpressing APP751. Menadione-induced production of reactive oxygen species was significantly decreased upon treatment with myricetin, quercetin or anthocyanin-rich extracts in a dose-dependent manner. However, these extracts did not affect caspase-3 activation, APP processing or Aβ levels upon staurosporine-induced apoptosis. APdE9 mice fed with anthocyanin-rich bilberry or blackcurrant extracts showed decreased APP C-terminal fragment levels in the cerebral cortex as compared to APdE9 mice on the control diet. Soluble Aβ40 and Aβ42 levels were significantly decreased in bilberry-fed mice as compared to blackcurrant-fed mice. Conversely, the ratio of insoluble Aβ42/40 was significantly decreased in blackcurrant-fed mice relative to bilberry-fed mice. Both berry diets alleviated the spatial working memory deficit of aged APdE9 mice as compared to mice on the control diet. There were no changes in the expression or phosphorylation status of tau in APdE9 mice with respect to diet. These data suggest that anthocyanin-rich bilberry and blackcurrant diets favorably modulate APP processing and alleviate behavioral abnormalities in a mouse model of AD.

Silver ion chromatography and gas chromatography-mass spectrometry in the structural analysis of cyclic dienoic acids formed in frying oils

The nature of the cyclic monoenoic fatty acids formed from linoleic acid in sunflower oil heated to 275°C has been determined by gas chromatography-mass spectrometry of the picolinyl ester derivatives, before and after hydrogenation and deuteration, and following simplification by silver ion high-performance liquid chromatography. In addition, they were examined by gas chromatography-Fourier transform infrared spectroscopy. Cyclopentene fatty acids (50% of the total monoenes) were formed from C-8 to C-12 and C-10 to C-14 of the original chain in equal amounts with unique stereochemistry. In some isomers the double bond appeared to remain in its original position, and in others it migrated to a substituted ring carbon. Isomers (25% of the total monoenes) were formed with a cis double bond in position 9 or 12 in equal amounts, and either a cyclopentane ring involving C-5 to C-9 or C-13 to C-17 or a cyclohexane ring involving C-5 to C-10 or C-12 to C-17 of the original fatty acid chain. With these compounds, most if not all of the possible configurational isomers were produced. Related compounds (25% of the total monoenes) with a trans double bond in position 9 or 12 were found with only six-membered rings with a restricted stereochemistry. Some bicyclic fatty acids were also present in the mixture.

Phytochemical Diversity in Tubers of Potato Cultivars and Landraces Using a GC-MS Metabolomics Approach

Phytochemical diversity with respect to a range of polar (including amino acids, organic acids, sugars, and sugar alcohols) and nonpolar (including fatty acids, alkanols, and sterols) metabolites was examined within tubers from a total of 29 genetically diverse potato cultivars and Chilean landraces using a metabolomics approach by gas chromatography-mass spectrometry. From principal component analysis of the polar and nonpolar metabolite data there was insufficient variation to differentiate the majority of cultivars and landraces. Analysis of all polar metabolite profiles revealed separation of two cultivars (Glenna and Morag) from the other cultivars and landraces and a separate cluster of one landrace line, largely due to higher levels of sugars. Pentland Javelin was distinct in containing high levels of many amino acids. The two Solanum tuberosum group phureja cultivars (Inca Sun and Mayan Gold) were not particularly similar and were not separated from the S. tuberosum group tuberosum cultivars. Analysis of the nonpolar metabolite data revealed partial separation of two landrace lines and, on the basis of some minor fatty acids, Mayan Gold was distinct. The differences in metabolite profiles are considered in terms of the taxonomy and breeding history of the cultivars and possible influences from other factors such as developmental stage of the tuber. With a view to exploring biosynthetic links between metabolites, a pairwise correlation analysis was performed on all metabolites. The significance of high correlations between many amino acids and between several nonpolar metabolites is discussed.

Polyphenol and vitamin C contents in European commercial blackcurrant juice products

Vitamin C and polyphenol contents (anthocyanins, proanthocyanidins, phenolic acids and flavonols) were analysed in commercial blackcurrant juice products purchased from various European countries (Finland, Poland, Germany, United Kingdom) using HPLC methods. The aim was to study variation between countries, as well as evaluate the intake of polyphenols from commercial juices. There was significant variation in the contents of polyphenols and vitamin C between countries. Expressed as the ready-to-drink beverages, German, Polish, Finnish and British products averaged anthocyanin contents of 38, 32, 12 and 7.5mg/2.5dl, proanthocyanidin contents of 27, 24, 10 and 1.2mg/2.5dl, flavonol contents of 16, 15, 5.2 & 1.9mg/2.5dl and phenolic acid contents of 12, 8.9, 3.7 and 1.5mg/2.5dl, respectively. The mean vitamin C content was highest in British (70mg/2.5dl) and lowest in Finnish products (15mg/2.5dl). The intake of polyphenols from German and Polish ready-to-drink beverages was clearly higher than that from Finnish, and especially, British beverages.

Spectroscopy and spectrometry of lipids — Part 1

For a collection of articles on spectroscopy and spectrometry of lipids to be comprehensive, there is a requirement for a wide range of instrumentation and applications to be considered. In this dossier, the aim is to include as many aspects as possible that are relevant to modern approaches in lipid analysis. It is hoped that the articles will show how enormous advances have been made, and are continuing to be made, in this area. The range of techniques includes infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (MS), ultraviolet (UV) spectroscopy and electron spin resonance (ESR) spectroscopy. IR spectroscopy can be subdivided further into mid- and near-infrared and Raman spectroscopy, and the former may be coupled to gas chromatography (GC). NMR spectroscopy encompasses high resolution (further split into 1 H, 13 C and 31 P) and time-domain. MS can be subdivided according to the type of ionisation (e.g. electron impact, chemical ionisation, fast atom bombardment) or separation technique (e.g. GC, liquid chromatography) to which it is coupled, but is more conveniently considered according to the type of lipid (fatty acid, triacylglycerol, complex polar lipid) to be analysed. Analysis of stable isotopes in fatty acids involves a highly specialised application of MS. There is a host of applications ranging from the measurement of broad quality control parameters to highly specific determinations for research purposes, and includes both qualitative and quantitative information. Quality control applications include chemical parameters such as peroxide value by IR (as a possible replacement for more timeconsuming traditional methods) and physical parameters such as droplet size distribution by time-domain NMR. Structural determinations of fatty acids and lipids by the various MS methods tend to be targeted towards specific research objectives.