Effects of fluorescent light on growth of bovine retinal pigment epithelial cells in vitro incubated with linoleic acid or linoleic acid hydroperoxide.

Light-induced peroxidation of polyunsaturated fatty acids (PUFA) may generate lipid hydroperoxides, which may have toxic effects on retinal pigment epithelial (RPE) cells in vitro. We investigated the effects of cool-white fluorescent light on the RPE cells incubated with linoleic acids (LA) or linoleic acid hydroperoxides (LHP) and the influence of antioxidative enzymes. We measured the bovine RPE cell number after exposure to fluorescent light (610 and 1,200 lux) in the presence of LA or LHP. Furthermore, the effects of superoxide dismutase (SOD) and catalase on LA- or LHP-treated RPE cells were also examined. Both LA and LHP treatment increased RPE cell number under weak illumination (610 lux), but dose-dependently decreased the number of cells exposed to strong illumination (1,200 lux). With exposure to strong illumination, LA caused a greater reduction in RPE cell number than LHP. Multiple linear regression analysis showed that the number of RPE cells was significantly decreased in a manner dependent on the interactions of the illuminance of light and the concentrations of LA or LHP. The antioxidative enzymes significantly ameliorated the damage to RPE cells from LA or LHP and exposure to light. Therefore, the exposure to fluorescent light augmented the cytotoxic effects of LA and LHP on RPE cells, and this effect is likely to be mediated by reactive oxygen species.     

Changes in Lipid Peroxide Level in Retinal Pigment Epithelial Cells In Vitro Upon Addition of Linoleic Acids or Linoleic Acid Hydroperoxides Under Varying Concentrations of Oxygen

We previously observed the presence of autofluorescent lipofuscin or its like in retinal pigment epithelial (RPE) cells, which were incubated with linoleic acid hydroperoxides (LHP). We studied the effect of oxygen on the level of lipid peroxides in RPE cells in the presence of linoleic acids (LA) or LHP. The level of lipid peroxides in these cells was determined by use of the thiobarbituric acid-reactive substance (TBARS), which responded to oxygen concentrations qualitatively, and a linear regression analysis. Multiple linear regression analysis disclosed that treatment with LA for 24 hr resulted in detectable increase in the level of TBARS in the cells, whereas treatment with LA or LHP for 48 hr caused detectable decrease. Stepwise linear regression analysis showed that the level of TBARS decreased in an oxygen-tension dependent manner in the cells incubated with LA for 48 hr. Thus, it was shown that short-term incubation with LA increased the level of TBARS in the cells and that LA decreased its level in an oxygen-tension dependent manner. For these results, the postulation was made that the prolonged auto-oxidation of LA caused production of lipofuscin-like materials, a complex of lipid peroxides and proteins that were insoluble in SDS and acetic acid solution.     

Peroxidation stimulated by lipid hydroperoxides on bovine retinal pigment epithelium mitochondria: effect of cellular retinol-binding protein

This study analyzes the effect of cellular retinol-binding protein (CRBP), partially purified from retinal pigment epithelium (RPE) cytosol, on the non-enzymatic lipid peroxidation induced by fatty acid hydroperoxides of mitochondrial membranes isolated from bovine RPE. The effect of different amounts (50, 75 and 100 nmol) of linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) on the lipid peroxidation of RPE mitochondria was studied; RPE mitochondria deprived of exogenously added hydroperoxide was utilized as control. The process was measured simultaneously by determining chemiluminescence as well as polyunsaturated fatty acid (PUFA) degradation of total lipids isolated from RPE mitochondria. The addition of hydroperoxides to RPE mitochondria produces a marked increase in light emission that was hydroperoxide concentration dependent. The highest value of activation was produced by LHP. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized RPE mitochondria incubated with and without hydroperoxides was found in the docosahexaenoic acid content, this decreased 40.90+/-3.01% in the peroxidized group compared to native RPE mitochondria. The decrease was significantly high: 86.32+/-2.57% when the lipid peroxidation was stimulated by 100 nmol of LHP. Inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (100-600 microg) of CRBP to RPE mitochondria. The inhibitory effect reaches the highest values in the presence of LHP.     

Effect of Linoleic Acid Concentration on Conjugated Linoleic Acid Production by Butyrivibrio fibrisolvens A38

Butyrivibrio fibrisolvens A38 inocula were inhibited by as little as 15 μM linoleic acid (LA), but growing cultures tolerated 10-fold more LA before growth was inhibited. Growing cultures did not produce significant amounts of cis-9, trans-11 conjugated linoleic acid (CLA) until the LA concentration was high enough to inhibit biohydrogenation, growth was inhibited, and lysis was enhanced. Washed-cell suspensions that were incubated anaerobically with 350 μM LA converted most of the LA to hydrogenated products, and little CLA was detected. When the washed-cell suspensions were incubated aerobically, biohydrogenation was inhibited, CLA production was at least twofold greater, and CLA persisted. The LA isomerase reaction was very rapid, but the LA isomerase did not recycle like a normal enzyme to catalyze more substrate. Cells that were preincubated with CLA lost their ability to produce more CLA from LA, and the CLA accumulation was directly proportional (r² = 0.98) to the initial cell density. Growing cells were as sensitive to CLA as LA, the LA isomerase and reductases of biohydrogenation were linked, and free CLA was not released. Because growing cultures of B. fibrisolvens A38 did not produce significant amounts of CLA until the LA concentration was high, biohydrogenation was arrested, and the cell density had declined, the flow of CLA from the rumen may be due to LA-dependent bacterial inactivation, death, or lysis.     

Some Analytical Observations of Autoxidation Products of Linoleic Acid and Their Thiobarbituric Acid Reactive Substances

Autoxidation products of linoleic acid (LA) were analyzed, when the weight became 1.14-fold under the autoxidation conditions of satisfactory atmospheric oxygen, at 37°C, in the dark, for 7 days. The LA absorbed 2.8 mol of oxygen to form secondary degradation products. This autoxidized LA consisted of 45% intact substance, 22% a mixture of polymers and endoperoxides, 18% LA hydroperoxides, 3% polar products, 1.7% azelaldehydeic acid, 1.3% hexahal, 0.9% azelaic acid, 0.6% octanoic acid, 0.3% suberaldehydeic acid, and so on. Thus, unstable 2,4-dienoic carbonyls were the main intermediate products of autoxidation of LA. Therefore, malonaldehyde was not a main product nor a major thiobarbituric acid reactive substance.     

Measurement of Binding of Ascorbic Acid to Myrosinase by Rate of Dialysis

The antioxidative activities of l-amino acids on the methylene blue sensitized photooxidation of linoleic acid (LA) were studied in a 40% aqueous ethanol solution at pH 7.0 and 33°C. His and CysH inhibited the photosensitized oxidation of LA, and the antioxidative activity of His was larger than that of CysH. The ratio of the chemical reaction rate constant for His and singlet oxygen (¹O₂) to that for LA and ¹O₂ was similar to the ratio of the ¹O₂ quenching rate constant for His to that for LA. This fact shows that His inhibits the photosensitized oxidation of LA by quenching ¹0₂ chemically.

On the other hand, CysH reacted with linoleic acid hydroperoxides (LAHPO) or with and caused the decomposition of LAHPO or the formation of Cys. This fact suggests that CysH inhibits the photosensitized oxidation of LA by decomposing LAHPO or by quenching ¹O₂ chemically.     

The Induction of Lipid Peroxidation in Rat Liver by Oral Intake of 9-Oxononanoic Acid Contained in Autoxidized Linoleic Acid

A component in the autoxidation products of linoleic acid (LA) which induced the endogenous lipid peroxidation was searched for. Secondary autoxidation products (SP) of LA induced the production of thiobarbituric acid reactive substances (TBARS) in liver as well as the LA hydroperoxides stimulated. SP were separated chromatographically into some fractions which were administered orally to rats. One fraction composed mainly of 9-oxononanoic acid (9-ONA) markedly stimulated the TBARS production in liver. Authentic 9-ONA also increased the lipid peroxide level as determined by a new test, as well as the activities of glutathione peroxidase and reductase in liver. Thus, hepatic lipid peroxidation was induced by orally given 9-ONA which was present in the autoxidation products of LA.     

Formation of Keto and Hydroxy Compounds of Linoleic Acid in Submitochondrial Particles of Bovine Heart

To observe lipid peroxidation of additive-free submitochondrial particles, we incubated submitochondrial particles in the absence of exogenous irons and t-butyl hydroperoxide. After the incubation, the phospholipids were hydrolyzed by phopholipase A₂, and the fatty acid constituents were analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry. Contrary to a commonly accepted theory, lipid peroxidation in the submitochondrial particles did not need the addition of NADH. In the phospholipid constituent fatty acids of the oxidized submitochondrial particles, derivatives of hydroperoxides of linoleic acid such as keto, hydroxy, trihydroxy, and hydroxyepoxy compounds were generated. Lipid peroxidation in the submitochondrial particles was not inhibited by the addition of catalase, superoxide dismutase, hydroxyl radical scavengers, or ethylenediaminetetraacetic acid, but was inhibited by the addition of KCN, antimycin-A, NADH, ubiquinol, deferoxamine mesylate, ascorbic acid, and -tocopherol. The cardiolipin–cytochrome c lipid peroxidation system could mimic the lipid peroxidation of the submitochondrial particles, in terms of linoleic acid products and the inhibitory patterns of radical scavengers and electron transfer chain inhibitors. Thus, lipid peroxidation in the submitochondrial particles seems to be due to phospholipid–hemoprotein lipid peroxidation systems such as the cardiolipin–cytochrome c system.     

Inactivation of Enzymes by Linoleic Acid Hydroperoxides and Linoleic Acid

The inactivation of the enzymes by linoleic acid hydroperoxides (LAHPO) was tested in connection with the toxicity of oxidized fat. At the same time, the inhibition of enzyme activities by linoleic acid was also tested. Ribonuclease (RNase), trypsin, chymotrypsin and pepsin which are considered to be simple proteins and not to be SH-enzymes were chosen as the enzymes. RNase was largely inhibited by LAHPO, but the other enzymes were inhibited by linoleic acid as well as LAHPO. The inhibition of each enzyme occurred at different pH. This fact may show that the inhibition occurs by binding of such hydrophobic compounds to the enzyme, and that the surface exposition of hydrophobic region may depend on the pH. Not only the reaction of some specific amino acid residue in the protein molecules with LAHPO, but also the binding of these hydrophobic compounds must be remembered in the mechanism of inhibition.     

Linoleic acid improves the robustness of cells in agitated cultures

The murine hybridoma (CC9C10) was subjected to high shear rates in a spinner flask to determine the effect of various culture additives on cell survival. At 500 rpm, the half-life of the viable cell concentration in a low protein serum-free medium was 50 min. Both bovine serum albumin and Pluronic F-68 had a significant effect in protecting cells under these conditions. The effects of the two supplements were additive, so that in the presence of both supplements there was minimal cell damage at 500 rpm. The survival rate of cells grown in media supplemented with linoleic acid improved significantly under high stirring rates. Cells grown for one passage in 50 μM linoleic acid and stirred at 500 rpm had a significantly higher survival rate than control cells. For cells grown over 5 passages in 25 μM linoleic acid, the survival rate at 470 rpm was ×3 greater than that determined for control cells. This difference gradually decreased at higher stirring rates up to 610 rpm when the half-life of the viable cell population was reduced to ∼10 min. Supplementation of cultures with linoleic acid has previously been shown to result in incorporation into all three cellular lipid fractions - polar, non-polar and free fatty acid (Butler et al., 1997). Our explanation for the increased survivability of the cells at high agitation rates in the presence of linoleic acid is that the structural lipid components of the cell including the outer membrane attained a higher unsaturated/saturated ratio which was more robust than that of control cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

气相色谱质谱联用技术对栝楼籽油亚麻酸和亚油酸含量的分析

通过皂化法和尿素包合法对热榨、冷榨栝楼（Trichosanthes kirilowii）籽油进行提取,采用气相色谱-质谱联用（GC-MS）技术检测分析栝楼籽油中亚麻酸、亚油酸成分、含量。结果表明,热榨栝楼籽油中亚麻酸相对含量为4.16%～11.58%,亚油酸相对含量为68.62%～95.84%。冷榨栝楼籽油中不含亚麻酸成分。此方法适用于栝楼籽油的成分分析。     

Comparative toxicity of oleic and linoleic acid on human lymphocytes

Commercially available lipid emulsions for parenteral nutrition are mainly composed by long chain triacylglycerol containing a high proportion of linoleic acid (LA) or oleic acid (OA). The immunological impact of such therapy is particularly important because parenteral diets are often administered to critically ill patients as a mechanism to supply adequate nutrition during catabolic stress conditions. The comparative toxicity of OA and LA on human lymphocytes and the type of cell death induced by these fatty acids were determined in vitro. Parameters of cell death were investigated by flow cytometry-cell viability, DNA fragmentation, phosphatidylserine externalization, mitochondrial depolarization, neutral lipid accumulation and production of reactive oxygen species-and by fluorescence microscopy-chromatin condensation. Additionally a spectrofluorometric assay was employed to determine the activities of caspase--3, 6 and 8. Evidence is presented herein that OA is less toxic to human lymphocytes than LA. However, both fatty acids promoted apoptosis and necrosis of these cells. The mechanism of cell death induced by OA involved activation of caspase 3 while the mechanism of death induced by LA involved mitochondrial depolarization and ROS production. Importantly, neutral lipid accumulation may be a mechanism to protect lymphocytes against the toxicity induced by OA. OA may offer an immunological less problematic alternative to LA with respect to fatty acid composition of parenteral nutritional emulsions.     

循环亚油酸水平与动脉粥样硬化风险的因果关联：一项两样本孟德尔随机化研究

摘要 目的：本文拟探讨遗传预测的循环亚油酸水平与不同部位动脉粥样硬化的因果关联。方法：采用两样本孟德尔随机化（Mendelian randomization, MR）研究方法,选择与亚油酸相关联的单核苷酸多态性位点（single nucleotide polymorphism, SNPs）作为工具变量（Instrument Variables, IVs）,评估遗传预测的循环亚油酸水平与不同部位动脉粥样硬化的因果关联。结果：逆方差加权法（Inverse Variance Weighted, IVW）分析结果显示,遗传预测的循环亚油酸水平与冠状动脉粥样硬化风险存在显著正相关（OR=1.32, 95% CI: 1.09-1.61, P=0.005）;循环亚油酸水平与脑动脉粥样硬化风险之间无因果关联 （OR=1.18, 95% CI: 0.63-2.23, P=0.602）。循环亚油酸水平与外周动脉粥样硬化风险存在显著负相关（OR= 0.55, 95% CI: 0.39-0.77, P=0.001）。循环亚油酸水平与其他动脉粥样硬化（不包括脑、冠状动脉和外周动脉）之间无显著的因果关联（OR=0.99, 95% CI: 0.81-1.21, P=0.916）。结论：遗传预测的循环亚油酸水平与冠状动脉粥样硬化及外周动脉硬化存在因果关联,亚油酸在动脉粥样硬化防治中的作用值得重视及进一步研究。     

An Evolutionary Perspective on Linoleic Acid Synthesis in Animals

The diet of organisms generally provides a sufficient supply of energy and building materials for healthy growth and development, but should also contain essential nutrients. Species differ in their exogenous requirements, but it is not clear why some species are able to synthesize essential nutrients, while others are not. The unsaturated fatty acid, linoleic acid (LA; 18:2n-6) plays an important role in functions such as cell physiology, immunity, and reproduction, and is an essential nutrient in diverse organisms. LA is readily synthesized in bacteria, protozoa and plants, but it was long thought that all animals lacked the ability to synthesize LA de novo and thus required a dietary source of this fatty acid. Over the years, however, an increasing number of studies have shown active LA synthesis in animals, including insects, nematodes and pulmonates. Despite continued interest in LA metabolism, it has remained unclear why some organisms can synthesize LA while others cannot. Here, we review the mechanisms by which LA is synthesized and which biological functions LA supports in different organisms to answer the question why LA synthesis was lost and repeatedly gained during the evolution of distinct invertebrate groups. We propose several hypotheses and compile data from the available literature to identify which factors promote LA synthesis within a phylogenetic framework. We have not found a clear link between our proposed hypotheses and LA synthesis; therefore we suggest that LA synthesis may be facilitated through bifunctionality of desaturase enzymes or evolved through a combination of different selective pressures.     

Linoleic acid-induced endothelial activation: role of calcium and peroxynitrite signaling

Hypertriglyceridemia, an important risk factor of atherosclerosis, is associated with increased circulating free fatty acids. Research to date indicates that linoleic acid (LA), the major fatty acid in the American diet, may be atherogenic by activating vascular endothelial cells. However, the exact signaling mechanisms involved in LA-mediated proinflammatory events in endothelial cells still remain unclear. We previously reported increased superoxide formation after LA exposure in endothelial cells. The objective of the present investigation is to determine the role of calcium and peroxynitrite in mediating the proinflammatory effect of LA in vascular endothelial cells. LA exposure increased intracellular calcium, nitric oxide, and tetrahydrodiopterin levels as well as the expression of E-selectin. Inhibiting calcium signaling using 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid and heparin decreased the expression of E-selectin. Also, LA-mediated nuclear factor kappa B activation and E-selectin gene expression were suppressed by Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (a superoxide scavenger), N(G)-monomethyl-l-arginine (an endothelial nitric oxide synthase inhibitor), and 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III) chloride (a peroxynitrite scavenger). LA exposure resulted in increased nitrotyrosine levels, as observed by Western blotting and immunofluorescence. Our data suggest that the proinflammatory effects of LA can be mediated through calcium and peroxynitrite signaling.     

龙生型高油酸花生种质油酸亚油酸含量及其比值的遗传分析

应用植物数量性状主基因+多基因混合遗传模型,对2个龙生型花生高油酸种质与低油酸珍珠豆型品种杂交组合F2的油酸、亚油酸含量及其比值(O/L值)进行遗传分析,结果表明:花生油酸、亚油酸含量的遗传均表现为1对主基因加性-显性模型。控制油酸含量主基因的加性、显性效应值和遗传率在组合I中分别为8.6281、-2.0164和65.26%,在组合II中则分别为10.6638、1.0652和71.39%;控制亚油酸含量主基因的加性、显性效应值和遗传率在组合I中分别为8.0327、1.2858和73.64%,在组合II中则分别为9.0885、-1.0826和71.59%。O/L值的遗传表现为2对主基因加性-显性-上位性模型。2对主基因的加性效应值分别为0.6855、0.6814(组合I)和1.6842、0.8835(组合II),显性效应值分别为-0.6838、0.024(组合I)和-1.6559、-0.5127(组合II);加性×加性效应(i)、加性×显性效应(jab)、显性×加性效应(jba)、显性×显性效应(l)分别为0.6812、0.024、-0.6803、-0.0244(组合I)和0.8822、-0.5124、-0.8594、0.496(组合II);组合I、II主基因遗传率分别为82.57%和88.64%。     

共轭亚油酸生理活性的作用机制

近年来共轭亚油酸的研究倍受关注,但其生理活性的作用机制还未得以充分阐明.从其所具备的各种生理活性的角度系统地综述了其可能的几种作用机制:作为抗氧化剂改变生物膜的结构,使其免受伤害;是致癌剂引发阶段的有效阻断剂;作用并激活过氧化物增生因子激活受体,从而调控基因的表达;影响某些细胞分裂素的合成和功能;影响生长激素的分泌;抑制恶性肿瘤细胞的增殖与黏附,恢复肿瘤细胞间的细胞间隙连接通讯功能;影响生物体内花生四烯酸和前列腺素的代谢.     

Effect of Linoleic Acid Hydroperoxides on Pepsin Activity

Pepsin was activated by incubating with linoleic acid hydroperoxides (LAHPO) at acidic region except at pH 4.0. Especially, the activation was best in pH 5.0 and 6.0 buffers, and the maximum activity attained was about 2 times of the original. It is still not certain whether LAHPO combined to the protein molecule or hydroperoxide groups reacted with some special amino acid residues in the protein molecule. The reactions were very sensitive to pH change and some conformational change of the protein molecule may be involved.     

Microencapsulation of Linoleic Acid with Low- and High-molecular-weight Components of Soluble Soybean Polysaccharide and Its Oxidation Process

Soluble soybean polysaccharide (SSPS) was fractionated into its low- (LMW) and high-molecular-weight (HMW) components to test their antioxidative and emulsifying properties. Linoleic acid was emulsified with an aqueous solution of SSPS, HMW, a mixture of LMW or HMW with maltodextrin, or maltodextrin alone. The emulsions prepared with SSPS, HWM and the mixture of HMW with maltodextrin were stable. These emulsions were spay-dried to produce microcapsules. The encapsulated linoleic acid was oxidized at 37°C and at various levels of relative humidity. Linoleic acid encapsulated with the mixture of LMW with maltodextrin or HMW was stable to oxidation, and this stability increased as the weight fraction of LMW in the mixture was increased. The LMW components also had high DPPH-radical scavenging activity. These results indicate that LMW played an important role in suppressing or retarding the oxidation of linoleic acid encapsulated with SSPS. The oxidative stability of linoleic acid encapsulated with a mixture of the LMW and HMW components was high at low and high relative humidity, but not at intermediate levels of relative humidity.