On the reaction of molecular oxygen with thiyl radicals: a re-examination

Absolute rate constants for the addition of oxygen to thiyl radicals, i.e. RS. + O2----RSOO., have been determined by applying a new competition method based on RS. formation via one-electron reduction of the corresponding disulphides, and the competition between RS. reacting with O2 and an electron donor such as ascorbate. Bimolecular rate constants have been obtained for the thiyl radicals derived from cysteine (6.1 X 10(7) mol-1 dm3 s-1), penicillamine (2.5 X 10(7) mol-1 dm3 s-1), homocysteine (8.0 X 10(7) mol-1 dm3 s-1), cysteamine (2.8 X 10(7) mol-1 dm3 s-1), 3-thiopropionic acid (2.2 X 10(8) mol-1 dm3 s-1) and glutathione (3.0 X 10(7) mol-1 dm3 s-1), respectively. The values obtained for the O2 addition to the thiyl radicals from glutathione and cysteine are considerable lower (by about two orders of magnitude) than those previously published. This indicates that the RS. + O2 reaction may be of complex nature and is generally a process which is not solely controlled by the diffusion of the reactants.     

Free radicals, polyunsaturated fatty acids, cell death, brain aging

Neuronal death generally involves, directly or indirectly, free radical attack and peroxidation. Targets are nucleic acids, proteins, the cytoskeleton, the extracellular matrix and especially membrane polyunsaturated fatty acids. a) One example for the fundamental role of fatty acids. Dietary fatty acids, and more particularly essential polyunsaturated fatty acids, have a direct influence on the composition of cerebral membranes, and hence on their functioning. Each of the two series of polyunsaturated fatty acids plays a particular role. In animals, a deficiency in linolenic acid causes serious perturbations in the nervous system. In fact, feeding animals with oils that have a low n-3 content leads to severe abnormalities in the composition of membranes, both of the brain and other organs. The rate of recovery from these anomalies is extremely slow in the brain, but rapid in the liver. Compared to certain other organs, the nervous system is neither protected against deficiency nor has it priority in the satisfaction if its needs. A decrease in acids of the linolenic series in the membranes results in a 40% reduction of Na-K-ATPase in nerve endings and a 20% reduction in 5'-nucleotidase. It also leads to anomalies in the electroretinogram which disappear with age. This deficiency in linolenic acid has little effect on motor function and disturbes activity and emotivity only slightly, but it seriously affects learning tasks. The presence of linolenic acid in the diet confers greater resistance to certain neurotoxic substances (triethyl lead, for example). Fatty acids essential for the brain could be those with very long chains. They are probably synthesized in the liver from linolenic and linoleic acids. They can also be supplied directly by food. However, if the diet contains a large proportion of very long chain fatty acids (fish oils), the lipid composition of all organs, including the brain, is altered. During the period of brain development there is a linear relation between the polyunsaturated fatty acid content of the brain and that of the diet. The requirement in linolenic acid is 200 mg/100 g of diet (0.4% of calories). That of linoleic acid is 1,200 mg/100 g of diet (2.4% of calories). b) Peroxidation of polyunsaturated fatty acids. Arachidonic acid is released by lysis of phospholipids (it is directly toxic), its peroxidized derivatives are extremely toxic. Peroxidation of membrane lipids alters enzymatic activity, the relationship between receptor and ligand, transport, and the symmetry of the lipid bilayer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Geometrical isomerism of monounsaturated fatty acids: thiyl radical catalysis and influence of antioxidant vitamins

Thiyl radicals generated either from thiols or disulfides act as the catalyst for the cis-trans isomerization of a variety of monounsaturated fatty acid methyl esters in homogeneous solution. Similar results have also been obtained using alpha-lipoic acid and its reduced form. The effectiveness of the isomerization processes in the presence of the most common antioxidants has been addressed. The ability of thiyl radical scavenging was found to increase along the series alpha-tocopherol < ascorbic acid < all-trans retinol. The cis-trans isomerization of fatty acid residues in multilamellar vesicles of dioleoyl phosphatidyl choline by thiyl radical, in the absence and presence of the various antioxidants, has also been studied in detail. The influence of the isomerization process on the phospholipid bilayer has been tested by permeability measurements of vesicles and it is clearly shown that trans fatty acid-containing membranes have intermediate properties between those formed by all-cis and saturated components. This study contributes to the understanding of radical processes that can alter or protect the naturally occurring cis geometry of unsaturated lipids in cell membranes and demonstrates a new role of essential antioxidants.     

Sulfinyl radical formation from the reaction of cysteine and glutathione thiyl radicals with molecular oxygen

Using Electron Spin Resonance spectroscopy at low temperatures, we find that thiyl radicals resulting from irradiation of frozen aqueous solutions of a variety of thiols, including cysteine, glutathione, and penicillamine react with oxygen to form sulfinyl (RSO.) radicals. The identity of the cysteine sulfinyl radical has been confirmed by the use of molecular oxygen isotopically labeled with 17O. Previous workers have suggested the reaction of thiyl radicals and molecular oxygen resulted in the formation of the potentially damaging thiol peroxyl radical, RSOO.; our work shows no evidence for this species. The sulfinyl radicals are suggested to result from a direct reaction between thiyl radicals and molecular oxygen. This reaction results in the cleavage of the dioxygen bond.     

Digestion and absorption of polyunsaturated fatty acids.

Polyunsaturated fatty acids play an important part in the structure and function of cellular membranes and are precursors of lipid mediators which play a key role in cardiovascular and inflammatory diseases. Dietary sources of essential fatty acids are vegetable oils for either linoleic or alpha-linolenic acids, and sea fish oils for eicosapentaenoic and docosahexaenoic acids. Because of the specificity of the pancreatic lipid hydrolases, triglyceride fatty acid distribution is an essential parameter in the digestibility of fats. The efficiency of the intestinal uptake depends on the hydrolysis and especially on their micellarization. n-3 polyunsaturated fatty acid ethyl ester digestion is recognized to be impaired, but n-3 polyunsaturated fatty acid triglyceride hydrolysis remains a controversial point, and to some authors explains differences observed between vegetable and fish oil absorption. So additional studies are required to investigate this intestinal step. In enterocytes, morphological and biochemical absorption processes involve reesterification of long-chain fatty acids and lipoprotein formation. At this level, specific affinity of I- and L-FABPc (cytosolic fatty acid binding proteins) to polyunsaturated fatty acids requires further investigation. A better understanding of the role of these FABPc might bring to light the esterification step, particularly the integration of polyunsaturated fatty acids into phospholipids. With reference to differences published between fish and vegetable oil absorption, longer-term absorption studies appear essential to some authors. Polyunsaturated fatty acid absorption is thought to be not very dissimilar to that of long-chain mono-unsaturated fatty acid absorption. However, several digestion and absorption specific steps are worth studying with reference to the crucial role of polyunsaturated fatty acids in the organism, and for example adaptation of possible dietary supplements.     

The influence of polyunsaturated fatty acids on probiotic growth and adhesion

The establishment of the intestinal microflora, and probiotic bacteria, may control the inflammatory conditions in the gut. As polyunsaturated fatty acids (PUFA) possess antimicrobial activities, they may deter the action of probiotics. We assessed whether free linoleic, gamma-linolenic, arachidonic, alpha-linolenic and docosahexaenoic acids at physiological concentrations in the growth media would influence the growth and adhesion of Lactobacillus GG (probiotic), Lactobacillus casei Shirota (probiotic) and Lactobacillus bulgaricus (dairy strain). Higher concentrations of PUFA (10-40 microg PUFA ml(-1)) inhibited growth and mucus adhesion of all tested bacterial strains, whilst growth and mucus adhesion of L. casei Shirota was promoted by low concentrations of gamma-linolenic acid and arachidonic acid (at 5 microg ml(-1)), respectively. PUFA also altered bacterial adhesion sites on Caco-2 cells. Caco-2 cells grown in the presence of arachidonic acid were less adhered to by all three bacterial strains. Yet, L. casei Shirota adhered better on Caco-2 cells grown in the presence of alpha-linolenic acid. As the adhesion to mucosal surfaces is pivotal in health promoting effects by probiotics, our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA.     

Interactions of thiyl free radicals with oxygen: a pulse radiolysis study

A pulse radiolysis study of glutathione in aqueous solution at pH 5.5 containing N2O/O2 mixtures at various ratios indicates that oxygen rapidly adds to the thiyl glutathione radical yielding a transient absorption, with a maximum at 540 nm, whose characteristics appear to be compatible with assignment to the GSOO. radical. The reaction (Formula: see text) appears to be an equilibrium whose kinetic constants have been estimated (kf = 2.0 X 10(9) dm3 mol-1, kb = 6.2 X 10(5) s-1). Evidence for electron transfer from ascorbate to the GSOO. radical has been obtained and the respective rate constant has been determined to be 1.75 +/- 0.15 X 10(8) dm3 mol-1 s-1.     

Influence of very-long-chain polyunsaturated fatty acids on membrane structure and dynamics

The unique attributes of very-long-chain polyunsaturated fatty acids (VLC-PUFAs), their long carbon chains (n > 24) and high degree of unsaturation, impart unique chemical and physical properties to this class of fatty acids. The changes imparted by VLC-PUFA 32:6 n-3 on lipid packing and the compression moduli of model membranes were evaluated from π-A isotherms of VLC-PUFA in 1,2-distearoyl-sn-3-glycero-phosphocholine (DSPC) lipid monolayers. To compare the attractive or repulsive forces between VLC-PUFA and DSPC lipid monolayers, the measured mean molecular areas (MMAs) were compared with the calculated MMAs of an ideal mixture of VLC-PUFA and DSPC. The presence of 0.1, 1, and 10 mol % VLC-PUFA shifted the π-A isotherm to higher MMAs of the lipids comprising the membrane and the observed positive deviations from ideal behavior of the mixed VLC-PUFA:DSPC monolayers correspond to repulsive forces between VLC-PUFAs and DSPC. The MMA of the VLC-PUFA component was estimated using the measured MMAs of DSPC of 47.1 ± 0.7 Å²/molecule, to be 15,000, 1100, and 91 Å²/molecule at 0.1, 1, and 10 mol % VLC-PUFA:DSPC mixtures, respectively. The large MMAs of VLC-PUFA suggest that the docosahexaenoic acid tail reinserts into the membrane and adopts a nonlinear structure in the membrane, which is most pronounced at 0.1 mol % VLC-PUFA. The presence of 0.1 mol % VLC-PUFA:DSPC also significantly increased the compression modulus of the membrane by 28 mN/m compared with a pure DSPC membrane. The influence of VLC-PUFA on lipid “flip-flop” was investigated by sum-frequency vibrational spectroscopy. The incorporation of 0.1 mol % VLC-PUFA increased the DSPC flip-flop rate fourfold. The fact that VLC-PUFA promotes lipid translocation is noteworthy as retinal membranes require a high influx of retinoids which may be facilitated by lipid flip-flop.     

The effects of n-3 and n-6 polyunsaturated fatty acids on plasma lipids and fatty acids of treated phenylketonuric children

Dietary-treated phenylketonuric patients (PKUs) display low levels of long-chain polyunsaturated fatty acids (PUFA) in plasma lipids. In a 6-month clinical trial we observed a decrease of triglycerides and an increase of n-3 long-chain PUFA in plasma of PKUs supplemented with fish oil, while no major differences in respect to the baseline values were found in a group supplemented with blackcurrant oil. A more complete source of long-chain PUFA of both the n-6 and n-3 series should be investigated for dietary supplementation of PKU patients.     

Long-chain polyunsaturated fatty acids, endothelial lipase and atherosclerosis

Endothelial lipase (EL), a new member of the lipase gene family, was recently cloned and has been shown to have a significant role in modulating the concentrations of plasma high-density lipoprotein levels (HDL). EL is closely related to lipoprotein and hepatic lipases both in structure and function. It is primarily synthesized by endothelial cells, functions at the cell surface, and shows phospholipase A1 activity. Overexpression of EL decreases HDL cholesterol levels whereas blocking its action increases concentrations of HDL cholesterol. Pro-inflammatory cytokines suppress plasma HDL cholesterol concentrations by enhancing the activity of EL. On the other hand, physical exercise and fish oil (a rich source of eicosapentaenoic acid and docosahexaenoic acid) suppress the activity of EL and this, in turn, enhances the plasma concentrations of HDL cholesterol. Thus, EL plays a critical role in the regulation of plasma HDL cholesterol concentrations and thus modulates the development and progression of atherosclerosis. The expression and actions of EL in specific endothelial cells determines the initiation and progression of atherosclerosis locally explaining the patchy nature of atheroma seen, especially, in coronary arteries. Both HDL cholesterol and EPA and DHA enhance endothelial nitric oxide (eNO) and prostacyclin (PGI2) synthesis, which are known to prevent atherosclerosis. On the other hand, pro-inflammatory cytokines augment free radical generation, which are known to inactivate eNO and PGI2. Thus, interactions between EL, pro- and anti-inflammatory cytokines, polyunsaturated fatty acids, and the ability of endothelial cells to generate NO and PGI2 and neutralize the actions of free radicals may play a critical role in atherosclerosis.     

Comparison of effects of dietary saturated, monounsaturated, and polyunsaturated fatty acids on plasma lipids and lipoproteins in man

Twenty patients consumed a liquid diet in which the predominant fatty acids were either saturated (Sat), monounsaturated (Mono), or polyunsaturated (Poly). The fats in these three diets comprised 40% of total calories and consisted of palm oil, high-oleic safflower oil, and high-linoleic safflower oil, respectively. During the third and fourth week of each dietary period, multiple samples of blood were taken and were analyzed for plasma total cholesterol (TC), triglycerides (TG), and cholesterol in lipoprotein fractions (VLDL-C, LDL-C, and HDL-C). Twelve of the patients had normal TG levels; in these patients, both Mono and Poly diets caused statistically significant and equal lowerings of plasma LDL-C, but the Poly diet lowered HDL-C levels more frequently than did the Mono diet. Neither diet changed the level of plasma TG. The proportions of total protein and the various lipid components in isolated fractions (VLDL, IDL, LDL, HDL) were not altered by the two diets. Eight patients had hypertriglyceridemia; these individuals showed considerable variability in response to Mono and Poly diets. Although there was a trend towards reductions in TC and LDL-C levels by both types of unsaturated fats, the changes were inconsistent; furthermore, HDL-C concentrations were low on the Sat diet and were unaffected by either the Mono or the Poly diet. The results of this study show that oleic acid is as effective as linoleic acid in lowering LDL-C levels in normo-triglyceridemic patients, and oleic acid seemingly reduces HDL-C levels less frequently than does linoleic acid. Neither type of unsaturated fat had striking effects on lipoprotein levels of hypertriglyceridemic patients.     

Bovine rod rhodopsin. 1. Bleaching by luminescence in vitro by recombination of radicals from polyunsaturated fatty acids

Rod outer segments of photoreceptors are characterized by rhodopsin, a membrane protein surrounded by phospholipids containing a very high concentration of polyunsaturated fatty acids. These fatty acids can propagate free radicals, initiated by peroxidation, whose recombination is eventually associated with light emission as chemiluminescence. The results reported here indicate that this effect produces an isomerization of the retinal (bleaching effect) of the rhodopsin, similar to that induced by light in normal vision. In vitro experiments on detergent-suspended rod outer segments (RdOS) from bovine eyes, using an enzymatic source of radicals, xanthine/xanthine oxidase, were carried out. The results indicate that the proposed mechanism is likely, because they can show the bleaching of rhodopsin in RdOS, owing to its extraordinary sensitivity. Thus this mechanism is, also, a possible explanation for anomalous visual effects such as light flashes (phosphene-like) perceived by humans. The functionality of the rhodopsin in the RdOS was first tested by visible light. Rhodopsin reactivation after bleaching was obtained by adding cis-retinal to the suspension, demonstrating the reversibility of the bleaching process. A special experimental system was developed to observe the bleaching from luminescence by radical recombination, avoiding physical contact between the rod outer segment suspension and the radicals to prevent radical-induced damage and modifications of the delicate structure of the rod outer segment.     

Changes in lipids containing long- and very long-chain polyunsaturated fatty acids in cryptorchid rat testes

The aim of the present study was to examine the effects of experimental cryptorchidism on rat testicular phospholipids and neutral lipids that contain long-chain (C(18)-C(22)) and very long-chain (VLC) (C(24)-C(32)) polyunsaturated fatty acids (PUFA). The weight of the cryptorchid testis was nearly half that of the contralateral control at postsurgical Days 7-10 owing to the depletion of germ cells. Concomitantly, the amounts of major glycerophospholipids (GPL) and sphingomyelin (SM) per testis decreased. Both these lipids lost their characteristic long-chain and very long-chain PUFA, notably 22:5n-6 and 28:4n-6, respectively, which suggests that these species are linked to the membranes of germ cells. In contrast, the amounts and concentrations of triglycerides (TG; triacylglycerols and 1-O-alkyl-2,3-diacylglycerols) and cholesterol esters (CE) increased several fold in the surviving cells (mainly Sertoli cells) in the cryptorchid testis. All these neutral lipids, but especially CE, accumulated large amounts of the major PUFA of the testis, 22:5n-6, as well as pentaenes with longer carbon chains (i.e., 24:5n-6 in TG and 28:5n-6 in CE). This accretion suggests that neutral lipids may store preformed PUFA coming from dying germ cell GPL and also VLCPUFA no longer needed as a source of PUFA destined to assemble new germ cell GPL. The lipid adjustments observed in cryptorchidism suggest a possible role for Sertoli cell CE in the turnover and conservation of PUFA within seminiferous tubules.     

Intravenous infusion of n-3 polyunsaturated fatty acids.

Dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) is regarded as beneficial for the prevention and treatment of atherosclerosis and thrombosis and chronic inflammatory diseases like rheumatoid arthritis and psoriasis. It may be possible to treat some acute diseases like acute myocardial infarction or acute rejection of grafted organs if it is possible to make n-3 PUFA take effect quickly (in hours instead of days). Three sets of experiments were done. In Experiment 1, emulsion of trieicosapentaenoyl-glycerol (EPA-TG) and tridocosahexaenoyl-glycerol was infused through rabbit ear veins, and the leukotriene B4/B5 production from polymorphonuclear leukocytes was measured at different time points by high-performance liquid chromatography. In Experiment 2, delayed type hypersensitivity (DTH) of mice was measured with sheep red blood cells as an antigen. Pure n-3 PUFA emulsions or a control solution were infused through tail veins just before the second challenge of the antigen. DTH was measured 24 hr after the second challenge. In Experiment 3, human natural killer cell activity was measured using K562 target cells before and after the infusion of pure EPA-TG emulsion to an antecubital vein. Leukotriene B4 production by rabbit polymorphonuclear leukocytes was depressed by 40% by EPA-TG infusion. DTH was suppressed almost completely by n-3 PUFA infusion. Natural killer cell activity was suppressed almost completely by EPA-TG infusion in 8 hr. DTH, natural killer cell activity, and leukotriene B4 production are probably related to acute rejection of grafted organs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dietary polyunsaturated fatty acids and the Pro12Ala polymorphisms of PPARG regulate serum lipids through divergent pathways: a randomized crossover clinical trial

Human and animal studies suggest an interaction between the Pro12Ala polymorphism of PPARG and dietary fat. In this randomized crossover clinical trial, we investigated whether subjects with the Pro12Pro and Ala12Ala genotypes of PPARG respond differently to a diet supplemented with high saturated (SAFA) or polyunsaturated fatty acid (PUFA).We recruited non-diabetic men from a population-based METSIM study (including 10,197 men) to obtain men with the Ala12Ala and the Pro12Pro genotypes matched for age and body mass index. Seventeen men with the Pro12Pro genotype and 14 with the Ala12Ala genotype were randomized to both a PUFA diet and a SAFA diet for 8 weeks in a crossover setting. Serum lipids and adipose tissue mRNA expression were measured during the diet intervention. At baseline, subjects with the Ala12Ala genotype had higher levels of HDL cholesterol and lower levels of LDL cholesterol, total triglycerides, and apolipoprotein B compared to those subjects with the Pro12Pro genotype (P < 0.05, FDR < 0.1). The Ala12Ala genotype also associated with higher mRNA expression of PPARG2, LPIN1, and SREBP-1c compared to participants with the Pro12Pro genotype (FDR < 0.001). On the other hand, PUFA diet resulted in lower levels of fasting glucose, total cholesterol, total triglycerides, and apolipoprotein B (P < 0.05, FDR < 0.1) but did not affect PPARG2 mRNA expression in adipose tissue. We conclude that individuals with the Pro12Pro genotype, with higher triglyceride levels at baseline, are more likely to benefit from the PUFA diet. However, the beneficial effects of dietary PUFA and the Ala12Ala genotype of PPARG on serum lipids are mediated through divergent mechanisms.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-015-0493-z) contains supplementary material, which is available to authorized users.     

Non-methylene-interrupted polyunsaturated fatty acids: effective substitute for arachidonate of phosphatidylinositol.

In mammalian tissues and cells, a characteristic of phosphatidylinositol (PI) is a high abundance of arachidonic acid (AA) relative to the other phospholipids. In this study, we investigated the effects of supplementation of several polyunsaturated fatty acids (PUFAs) on the AA concentration of the PI fraction using a cultured cell system. Neither alpha-linolenic acid nor eicosapentaenoic acid supplement reduced the level of AA in PI of HepG2 cells. In contrast to the n-3 series PUFAs, adding podocarpic acid (20:3, Delta-5,11,14) and pinolenic acid (18:3, Delta-5,9,12) reduced the AA content of the PI fraction from a control value of 15.9% to 7.0 and 8.7%, respectively. In the experiments with pinolenic acid, selective and significant accumulation of 20:3 (Delta-7,11,14), the chain-elongated metabolite of pinolenic acid, was observed in the PI fraction. On the other hand, adding columbinic acid (18:3, Delta-5t,9,12) had no effect on AA content of the PI fraction. Because both podocarpic acid and pinolenic acid are non-methylene-interrupted fatty acids (NMIFAs) that are not converted to AA metabolically, these NMIFAs may be interesting experimental tools for research on the function of PI-origin bioactive lipids.