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The primary products from peroxidation of linoleate in biological tissues and fluids are the hydroperoxy octadecadienoates, and the products normally assayed, after reduction of the hydroperoxides, are the corresponding hydroxy octadecadienoates (HODEs). The HODEs are found in tissues and fluids as a mixture of Z,E and E,E stereoisomers. Two regioisomeric sets of Z,E and E,E stereoisomers are normally observed with substitution at the 9- and 13-positions of the 18-carbon chain. The Z,E/E,E product ratio has proved to be a useful means for assessing the reducing capacity of the medium undergoing peroxidation. The HODE Z,E/E,E product ratios previously reported for tissues such as liver and brain vary from 0.5 to 2.0, and plasma ratios are somewhat higher, between 2.0 and 3.0. The reported literature protocols for HODE assay in tissues involve homogenization, reduction with sodium borohydride in the presence of BHT, and ester hydrolysis with KOH to give the free HODEs. This is followed by either reverse-phase HPLC of the free acid HODEs or by conversion to TMS derivatives and GC-MS. When sodium borohydride is replaced in the protocol by triphenylphosphine, a gentler reducing agent, HODE Z,E/E,E product ratios are much higher, and lower total HODE levels of are found. It is proposed that inclusion of sodium borohydride in the isolation procedures leads to ex vivo reactions that are avoided if triphenylphosphine is used as the reducing agent. Modified protocols for HODE analyses (tissue and plasma methods #2) are described that should be used for assays of tissues and fluids.
The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) causes a rapid reduction of body and adipose mass in mice. In addition to changes in adipose tissue, numerous studies have reported alterations in hepatic lipid metabolism. Livers of CLA-fed mice gain mass, partly due to lipid accumulation; however, the precise molecular mechanisms are unknown. To elucidate these mechanisms, we examined fatty acid composition and gene expression profiles of livers from a polygenic obese line of mice fed 1% trans-10, cis-12-CLA for 14 days. Analysis of gene expression data led to the identification of 1393 genes differentially expressed in the liver of CLA-fed male mice at a nominal P value of .01, and 775 were considered significant using a false discovery rate (FDR) threshold of .05. While surprisingly few genes in lipid metabolism were impacted, pathway analysis found that protein kinase A (PKA) and cyclic adenosine monophosphate (cAMP) pathways signaling pathways were affected by CLA treatment and 98 of the 775 genes were found to be regulated by hepatocyte nuclear factor 4alpha, a transcription factor important in controlling liver metabolic status.
Copyright 2010 Elsevier Inc. All rights reserved.
The objective of this study was to determine whether increasing levels of dietary safflower oil would alter unsaturated fat (especially CLA) and tocopherol content of lamb, animal performance, carcass characteristics, or color stability of lamb muscle tissue. Targhee x Rambouillet wethers (n = 60) were assigned to one of three diets (four pens per treatment with five lambs per pen) in a completely random design. Diets were formulated with supplemental safflower oil at 0 (control), 3, or 6% (as-fed basis) of the diet. Diets containing approximately 80% concentrate and 20% roughage were formulated, on a DM basis, to be isocaloric and isonitrogenous and to meet or exceed NRC requirements for Ca, P, and other nutrients. A subsample of 12 wethers per treatment was selected based on average BW (54 kg) and slaughtered. Carcass data (LM area, fat thickness, and internal fat content) and wholesale cut weight (leg, loin, rack, shoulder, breast, and foreshank), along with fatty acid, tocopherol, and color analysis, were determined on each carcass. The LM and infraspinatus were sampled for fatty acid profile. Increasing safflower oil supplementation from 0 to 3 or 6% increased the proportion of linoleic acid in the diet from 49.93 to 55.32 to 62.38%, respectively, whereas the percentage of oleic acid decreased from 27.94 to 23.80 to 20.73%, respectively. The percentage of oil in the diet did not (P > or = 0.11) alter the growth and carcass characteristics of lambs, nor did it alter the tocopherol content or color stability of meat. Increasing levels of safflower oil in lamb diets decreased (P < 0.01) the weight percentage of oleic acid in the infraspinatus and LM, and increased linoleic acid (P < 0.01). Oil supplementation increased (P < 0.01) the weight percentage of various isomers of CLA in muscle, with the greatest change in the cis-9,trans-11 isomer. Supplementation of sheep diets with safflower oil, up to 6% of the diet, resulted in increasing levels of unsaturated fatty acids and CLA in the lean tissue, without adversely affecting growth performance, carcass characteristics, or color stability of lamb.
The reported crystal structures of plant and animal lipoxygenases (LOX) show that the nonheme iron in the catalytic domain is ligated by three histidines, the C-terminal isoleucine, and in certain structures also by a fifth iron ligand, an asparagine or histidine residue. Mouse 8-LOX and its homologues (e.g., human 15-LOX-2) are unique in having a serine in place of the usual Asn or His in this fifth position. To investigate the importance of the residue in mouse 8-LOX structure-function, the serine-558 was replaced by asparagine, histidine, or alanine using oligonucleotide-directed mutagenesis. Wild-type mouse 8-LOX and the mutant cDNAs were expressed in HeLa cells infected with vaccinia virus encoding T7 RNA polymerase and their relative lipoxygenase activities assessed by incubation with [14C]arachidonic acid or [14C]linoleic acid followed by HPLC analysis of the products. The Ser558Asn and Ser558His mutants had equivalent or greater activity than wild-type 8-LOX. They also exhibited some 15-LOX activity, indicating that small structural perturbations (in this case to a residue identical in mouse 8-LOX and its 15-LOX-2 homologues) can interchange the positional specificity of these closely related enzymes. Remarkably, the Ser558Ala mutant exhibited significant 8-LOX activity, indicating that this position is not an essential iron ligand in the enzyme. We conclude that mouse 8-LOX is catalytically competent with only four amino acid iron ligands, and that Ser-558 of the wild-type enzyme does not play an essential role in catalysis.
Oxidative modification of LDL is believed to play a major role in atherogenesis. As major lipid peroxidation products oxygenated linoleic acid derivatives and oxysterols have been described in human atherosclerotic lesions. Here we report that human lesions contain isoprostanes as peroxidation products of arachidonic acid at a level of 27.1 +/- 21.2 pg/mg wet weight (n = 10), which corresponds to 75.9 +/- 59.3 pg/mg dry weight, n contrast, human umbilical veins (n = 10), which were used as nonatherosclerotic control vessels, contain much smaller amounts of isoprostanes (1.4 +/- 0.7 pg/mg wet weight, which corresponds to 11.7 +/- 6.2 pg/mg dry weight), and there are significant differences between the two types of vessels. As major products of linoleic acid oxidation, racemic hydroxy linoleate isomers were detected in the lesional ester lipids. In human lesions, the hydroxy linoleic acid/linoleic acid ratio was about 0.5%, a result indicating that 5 out of 1000 linoleate residues are present as hydroxylated derivatives. In umbilical veins, no hydroxy linoleic acid could be detected. These data show that human atherosclerotic lesions contain increased amounts of hydroxy linoleic acid isomers and isoprostanes when compared with nonatherosclerotic vessel wall and suggest a link between local lipid peroxidation and progression of atherosclerosis. For evaluation of the degree of lipid peroxidation, the determination of the hydroxy linoleic acid/linoleic acid ratio appears to be more suitable than the isoprostane content.
We have investigated lipid peroxidation in the skin of CD1 mice following single or repeated topical applications of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). A substantial accumulation of hydroxyphospholipids, to levels 3-5 times control values, followed exposure to two or more TPA treatments (24-72 h intervals), whereas single applications were ineffective. Sodium borohydride reduction increased the yield of product by approximately 50%, suggesting the additional presence of phospholipid hydroperoxides in the oxidized lipids. Straight phase HPLC analysis of the constituent hydroxy fatty acids, followed by gas chromatography/mass spectrometry, revealed that oxidized derivatives of linoleic acid, including 9- and 13-hydroxyoctadecadienoic acids (9- and 13-HODE), were the primary products. Stereochemical analysis showed ratios of S to R stereoisomers of 1.3 for 13-HODE and 1.27 for 9-HODE, which implied that TPA-induced peroxidation was primarily due to free radical oxidation, although a partial contribution of enzyme (lipoxygenase) activity is possible. The TPA-induced peroxidation was greater in the epidermis than in the dermis. Pre-exposure of mouse skin to the anti-inflammatory agent fluocinolone acetonide, antioxidants and enzyme (phospholipase A2 and lipoxygenase) inhibitors lowered the peroxidation response to subsequent exposure to TPA. Phospholipid peroxidation products may be useful markers of oxygen radical production in TPA-exposed mouse skin with possible relevance to tumor promotion.