linoleic-acid and parinaric-acid

The influence of the polyunsaturated fatty acid (PUFA) composition of the diet on the rate of fatty acid turnover of individual phospholipids in the erythrocyte membrane in vivo was studied. Following modification of the fatty acid composition of the membrane phospholipids by the use of a fish oil or a linoleic acid enriched diet, phospholipids--labelled in the unsaturated fatty acid at the 2-position of the glycerol moiety--were introduced into the membrane of freshly isolated rabbit erythrocytes. Thereafter, the labelled erythrocytes were reinjected into the bloodstream of the animal. It appears that, with the exception of 1-palmitoyl,2-linoleoyl phosphatidylcholine, all other phosphatidylcholines disappear faster from the erythrocytes of fish oil-fed rabbits than from the red cells of linoleic acid-fed rabbits. Another parameter, which possibly influences the turnover rates of PUFA containing phospholipids, can be peroxidation. An attempt was made to measure peroxidative damage of lipids in vivo by the introduction of 1-palmitoyl,2-cis-parinaroyl phosphatidylcholine (PnPC)--a probe to measure oxidative stress--into the membrane of freshly isolated erythrocytes, in the same way as is described for the radioactive phospholipids. The data demonstrate that the fluorescent signal from the PnPC decreases at a fast rate which is independent of the dietary conditions.

Topics: Animals; Cell Membrane; Diet; Erythrocytes; Fatty Acids; Fatty Acids, Unsaturated; Female; Fish Oils; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Membrane Lipids; Phosphatidylcholines; Phosphatidylethanolamines; Phospholipids; Rabbits; Vitamin E

This study has investigated the effect of dietary vitamin E on markers of antioxidant status. Four groups of rabbits received diets containing 30 energy percent (en%) total fat (7.8 en% contributed by linoleic acid) for 12 weeks. D,1-alpha tocopheryl acetate was added to the diets to obtain a range of vitamin E concentrations (49, 114, 179, or 775 tocopherol equivalents per kg diet). Increased vitamin E concentrations were demonstrated in plasma lipoproteins and erythrocyte membranes following supplementation, and dietary effects on lipid peroxidation were investigated by (i) monitoring a fluorescent parinaric acid probe incorporated into erythrocyte membranes in vivo, (ii) determination of malondialdehyde and oxysterols in plasma, and (iii) investigation of the susceptibility of low density lipoprotein (LDL) to copper-induced conjugated diene formation in vitro. No effects of vitamin E were observed on parinaric acid oxidation in vivo or on the accumulation of lipid peroxidation products in plasma, but the resistance of LDL to oxidation in vitro increased significantly as vitamin E was supplemented to the diets. Our results demonstrate that under these dietary conditions (7.8 en% linoleic acid) increasing the vitamin E content of plasma and erythrocytes approximately two-fold does not reduce the level of lipid peroxidation in vivo, indicating sufficient antioxidant capacity on the lowest vitamin E diet. In contrast, LDL became more resistant to an extreme oxidative stress applied in vitro. The relevance of these assays to currently proposed mechanisms of atherosclerosis is discussed.

Topics: alpha-Tocopherol; Animals; Antioxidants; Cholesterol; Diet; Dietary Fats; Erythrocyte Membrane; Fatty Acids, Unsaturated; Fluorescent Dyes; Linoleic Acid; Linoleic Acids; Lipid Peroxidation; Lipids; Lipoproteins; Lipoproteins, LDL; Oxidation-Reduction; Phospholipids; Rabbits; Regression Analysis; Spectrometry, Fluorescence; Tocopherols; Triglycerides; Vitamin E

Methanol extracts of the hepatopancreas of mussels (Mytilus edulis) harvested at two locations (Ship Harbour and Wine Harbour) in eastern Nova Scotia, Canada, were found to be toxic to mice after intraperitoneal injection. The commonly known toxins, such as those associated with diarrhetic shellfish poison (DSP), paralytic shellfish poison, and domoic acid, were not present in the extracts. However, they were found to contain elevated levels of free fatty acids. Using a modified DSP extraction procedure the quantities of free fatty acids determined (by latroscan TLC/FID) in the hepatopancreases of mussels were 2.9 mg/g (Ship Harbour 1), 2.2 mg/g (Ship Harbour 2), 1.2 mg/g (Wine Harbour), and 0.15 mg/g (Prince Edward Island, control). After further investigation it was determined that certain unsaturated fatty acids were mainly responsible for the toxicity. These included palmitoleic, linoleic, linolenic, octadecatetraenoic, and eicosapentenoic acids. Artificial mixtures of pure standards of these acids prepared in the same concentrations as found in the shellfish samples were also toxic to mice. These results indicate that elevated levels of free fatty acids in mussel hepatopancreas from locations in eastern Canada can lead to mouse deaths when using the DSP mouse bioassay procedure.

Topics: alpha-Linolenic Acid; Animals; Bivalvia; Chromatography, Thin Layer; Eicosapentaenoic Acid; Fatty Acids, Monounsaturated; Fatty Acids, Nonesterified; Fatty Acids, Unsaturated; Flame Ionization; Glycerides; Injections, Intraperitoneal; Linoleic Acid; Linoleic Acids; Lipid Metabolism; Lipids; Male; Mice; Nova Scotia

The enzyme delta 6-desaturase is responsible for the conversion of linoleic acid (18:2) to gamma-linolenic acid (18:3 gamma). A cyanobacterial gene encoding delta 6-desaturase was cloned by expression of a Synechocystis genomic cosmid library in Anabaena, a cyanobacterium lacking delta 6-desaturase. Expression of the Synechocystis delta 6-desaturase gene in Anabaena resulted in the accumulation of gamma-linolenic acid (GLA) and octadecatetraenoic acid (18:4). The predicted 359 amino acid sequence of the Synechocystis delta 6-desaturase shares limited, but significant, sequence similarity with two other reported desaturases. Analysis of three overlapping cosmids revealed a delta 12-desaturase gene linked to the delta 6-desaturase gene. Expression of Synechocystis delta 6- and delta 12-desaturases in Synechococcus, a cyanobacterium deficient in both desaturases, resulted in the production of linoleic acid and gamma-linolenic acid.

Topics: Amino Acid Sequence; Anabaena; Base Sequence; Cloning, Molecular; Conjugation, Genetic; Cyanobacteria; Fatty Acid Desaturases; Fatty Acids, Unsaturated; gamma-Linolenic Acid; Genes, Bacterial; Genetic Linkage; Linoleic Acid; Linoleic Acids; Linolenic Acids; Linoleoyl-CoA Desaturase; Molecular Sequence Data; Protein Structure, Secondary; Transformation, Genetic

Intestinal enterocytes contain two homologous fatty acid-binding proteins, intestinal fatty acid-binding protein (I-FABP)2 and liver fatty acid-binding protein (L-FABP). Since the functional basis for this multiplicity is not known, the fatty acid-binding specificity of recombinant forms of both rat I-FABP and rat L-FABP was examined. A systematic comparative analysis of the 18 carbon chain length fatty acid binding parameters, using both radiolabeled (stearic, oleic, and linoleic) and fluorescent (trans-parinaric and cis-parinaric) fatty acids, was undertaken. Results obtained with a classical Lipidex-1000 binding assay, which requires separation of bound from free fatty acid, were confirmed with a fluorescent fatty acid-binding assay not requiring separation of bound and unbound ligand. Depending on the nature of the fatty acid ligand, I-FABP bound fatty acid had dissociation constants between 0.2 and 3.1 microM and a consistent 1:1 molar ratio. The dissociation constants for L-FABP bound fatty acids ranged between 0.9 and 2.6 microM and the protein bound up to 2 mol fatty acid per mole of protein. Both fatty acid-binding proteins exhibited relatively higher affinity for unsaturated fatty acids as compared to saturated fatty acids of the same chain length. cis-Parinaric acid or trans-parinaric acid (each containing four double bonds) bound to L-FABP and I-FABP were displaced in a competitive manner by non-fluorescent fatty acid. Hill plots of the binding of cis- and trans- parinaric acid to L-FABP showed that the binding affinities of the two sites were very similar and did not exhibit cooperativity. The lack of fluorescence self-quenching upon binding 2 mol of either trans- or cis-parinaric acid/mol L-FABP is consistent with the presence of two binding sites with dissimilar orientation in the L-FABP. Thus, the difference in binding capacity between I-FABP and L-FABP predicts a structurally different binding site or sites.

Topics: Carrier Proteins; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Fatty Acids; Fatty Acids, Unsaturated; Intestinal Mucosa; Kinetics; Linoleic Acid; Linoleic Acids; Liver; Mathematics; Neoplasm Proteins; Nerve Tissue Proteins; Oleic Acid; Oleic Acids; Recombinant Proteins; Spectrometry, Fluorescence; Stearic Acids

Release of superoxide from human neutrophils was stimulated by cis-unsaturated fatty acids (e.g. arachidonate, linoleate). All the saturated and trans-unsaturated fatty acids tested were ineffective in this context. Binding of linoleate to neutrophils was biphasic and could be resolved into a linear and a saturable component. The extent of linoleate binding to the saturable component correlated strongly with the amount of O-2 released (r = 0.96). Palmitate, a saturated fatty acid, exhibited only linear binding to neutrophils and the binding was similar to the linear component of linoleate binding. All cis-unsaturated fatty acids tested decreased the fluorescence polarization of cis- and trans-parinaric acid used as membrane probes in suspensions of neutrophils, whereas the saturated fatty acid, myristate, increased the polarization. Fatty acids which stimulated O-2 release induced morphological changes (i.e. evaginations) in neutrophils, whereas the inactive fatty acids did not affect the cellular morphology. Effects on both the cell morphology and superoxide release were reversed by treatment of the cells with delipidated albumin. These data are discussed in relation to recent reports concerning the effects of free fatty acids on various biochemical systems.

Topics: Arachidonic Acid; Arachidonic Acids; Fatty Acids, Nonesterified; Fatty Acids, Unsaturated; Fluorescent Dyes; Hot Temperature; Humans; Linoleic Acid; Linoleic Acids; Neutrophils; Palmitic Acid; Palmitic Acids; Serum Albumin; Superoxides; Time Factors