Free radicals induced peroxidations and isomerizations of unsaturated fatty acids

Uzročno-posljedične veze između slobodnih radikala u biološkim sustavima i različitih patoloških stanja omogućavaju da se radijacijsko-kemijske metode koriste za pripravu i praćenje njihovih reakcija u kontroliranim uvjetima. U ovom su radu istraživani radikali višenezasićene masne kiseline, prekursori za dvije vrste procesa, peroksidaciju i, posebno u prisustvu biološki važnih tiola, cis, trans-izomerizaciju na dvostrukoj vezi linolne kiseline, LiH. Pritom je razvijen modelni sustav micela LiH u kojemu se radikalski procesi mogu selektivno pratiti u uvjetima koji se, za razliku od fizioloških uvjeta, mogu prilagođavati potrebama eksperimentiranja. Prvi put se istražila povezanost tih dvaju radikalima induciranih procesa u biomimetičkim uvjetima te je pokazano da hidroperoksidi (LiOOH) i transkonfiguracija LiH nastaju i u oksidacijskim uvjetima. Biomimetički model priređen je s dodatkom 2-merkaptoetanola kao amfifilnog tiola, a inicijacija radikalskih procesa provedena gama zračenjem s dozama zračenja do 400 Gy u različitim uvjetima. U uvjetima ravnoteže sa zrakom uz značajnu koncentraciju LiOOH (~105 mol dm3) odvijao se i proces cis, transizomerizacije s katalitičkim ciklusom 370. Kako bi se predvidio utjecaj samoorganiziranih supramolekulskih struktura LiH na ispitivane procese istražen je i utjecaj veličine micela te prisutnost prirodnih antioksidansa.The cause-effect relationship between free radicals in biological systems and the various pathological conditions enable us to use the radiation-chemical methods for preparation and determination these reactions under controlled conditions. In this paper we investigated polyunsaturated fatty acid radicals which are precursors for the two types of processes, peroxidation and, especially in the presence of biologically relevant thiol, cis, transisomerization of linoleic acid (LiH). A model micellar system was developed in which radical processes of LiH were selectively determined under conditions that, in contrast to the physiological, could be adapted to the experiments requirements. These two radical-induced processes were compared for the first time under biomimetic conditions demonstrating that hydroperoxides (LiOOH) and trans-lipids can be formed under oxidizing conditions. Biomimetic model was prepared with the addition of 2-mercaptoethanol as amphiphilic thiol, and radical processes initiation was performed by -radiation up to 400 Gy under different conditions. In air-equilibrated solutions, together with substantial amount of LiOOH (~10-5 mol dm3), the cis, trans-isomerization process was also observed with a catalytic cycle of 370. In order to envisage the impact of self-organized supramolecular organization of LiH on the outcome of lipid peroxidation and cis, trans-isomerization processes, the influence of the micelle size and the presence of natural antioxidants were also studied

The influence of antioxidants in the thiyl radical induced lipid peroxidation and geometrical isomerization in micelles of linoleic acid

The biomimetic model of micelles of linoleic acid containing 2-mercaptoethanol and the antioxidant was examined under gamma irradiation up to 400 Gy in aerobic or deoxygenated conditions where thiyl radicals are the main reactive species. Lipid peroxidation was retarded by ascorbic acid and α-tocopherol, whereas this process was strongly inhibited by resveratrol as effectively as the ascorbic acid/α-tocopherol mixture. Furthermore, antioxidants have a much stronger inhibitory effect on the peroxidation in the presence of 2-mercaptoethanol, and at the same time show protective properties of the double bond, decreasing the cis–trans isomerization. Under anaerobic conditions cis–trans isomerization occurred and antioxidants efficiency increased along the series: resveratrol α-tocopherol ascorbic acid. This result is explained taking into account the double bond localization in the hydrophobic core of the micelle and the need of co- localization of the antioxidant in order to get an anti-isomerizing activity and protection of the natural lipid geometry

Application of 4-amino-N-adamantylphthalimide solvatochromic dye for fluorescence microscopy in selective visualization of lipid droplets and mitochondria

4-Amino-N-adamantylphthalimide (1) is a dye with moderate absorptivity (in CH3CN ε363 = 4200 M-1 cm-1) and high quantum yield of fluorescence (ΦF = 0.15-0.80) that exhibits fluorosolvatochromic properties. The dye can be excited at 405 nm and the position of fluorescence maximum and the Stokes shift are well correlated with the ET(30) parameter. The excitation in the near-visible part of the spectrum and low cytotoxicity allow use of the dye in live cell microscopy. Due to its amphiphilic character, the dye stains artificial membranes in liposomes. Using confocal microscopy on two human cancer cell lines, we have shown that 1 stains primarily intracellular lipid droplets. Colocalization experiments with different organelle markers indicated that 1 additionally stains mitochondrial membranes. The fluorosolvatochromism of 1 allows the simultaneous visualization of mitochondria and intracellular lipid droplets in two separate emission channels, which has a potential use in cells and tissues exhibiting intense oxidative metabolism of lipids

Leukocyte Activation and Antioxidative Defense Are Interrelated and Moderately Modified by n-3 Polyunsaturated Fatty Acid-Enriched Eggs Consumption—Double-Blind Controlled Randomized Clinical Study

This placebo-controlled, double-blind, randomized, interventional study investigated the effects of low/intermediate doses of n-3 polyunsaturated fatty acids (PUFAs) on the endothelial function, markers of leukocyte activation, and oxidative status following dietary intake of n-3 PUFA-enriched hen eggs in young healthy individuals. Twenty young healthy adults of both sexes who consumed n-3 PUFA- enriched hen eggs (two eggs per day, for three weeks, total of approximately 407 mg/day n-3 PUFAs) or regular eggs (two eggs per day for three weeks, total of approximately 75 mg/day n-3 PUFAs) participated in this study. Skin microvascular endothelium-independent and endothelium-dependent vasodilation were assessed by laser Doppler flowmetry. Serum lipid profile and content of free fatty acids, markers of leukocyte activation, biochemical parameters of oxidative stress, as well as antioxidative enzymes serum activity were measured before and after respective dietary protocol. The results of this study revealed significant differences in the markers of leukocyte activation (such as CD11a/LFA-1) and antioxidative defense, which are related to increased intake of n-3 PUFAs, providing the evidence that consumption of nutritionally enriched hen eggs may affect physiological processes related to oxidative balance. The absence of significant changes in microvascular reactivity following supplementation with a low-intermediate dose of n-3 PUFAs, unlike in our previous studies where functional eggs contained ~1 g of n-3 PUFA, suggests the existence of a dose-dependent effect

Trefoil Factor 3 Deficiency Affects Liver Lipid Metabolism

Background/Aims: Tff3 protein plays a well recognized role in the protection of gastrointestinal mucosa. The role of Tff3 in the metabolism is a new aspect of its function. Tff3 is one of the most affected liver genes in early diabetes and fatty liver rodent models. The aim of this study was to investigate the effect of Tff3 deficiency on lipid and carbohydrate metabolism and on markers of oxidative stress that accompanies metabolic deregulation. Methods: Specific markers of health status were determined in sera of Tff3 deficient mice, including glucose level, functional glucose and insulin tolerance. Composition of fatty acids (FAs) was determined in liver and blood serum by using gas chromatography. Oxidative stress parameters were determined: lipid peroxidation level via determination of lipid hydroperoxide and thiobarbituric acid reactive substances (TBARS), antioxidative capacity (FRAP) and specific antioxidative enzyme activity. The expression of several genes and proteins related to the metabolism of lipids, carbohydrates and oxidative stress (CAT, GPx1, SOD2, PPARα, PPARγ, PPARδ, HNF4α and SIRT1) was determined. Results: Tff3 deficient mice showed better glucose utilization in the glucose and insulin test. Liver lipid metabolism is affected and increased formation of small lipid vesicles is noticed. Formation of lipid droplets is not accompanied by increased liver oxidative stress, although expression/activity of monitored enzymes is deregulated when compared with wild type mice. Tff3 deficient mice exhibit reduced expression of metabolism relevant SIRT1 and PPARγ genes. Conclusion: Tff3 deficiency affects the profile and accumulation of FAs in the liver, with no obvious oxidative stress increase, although expression/activity of monitored enzymes is changed as well as the level of SIRT1 and PPARγ protein. Considering the strong downregulation of liver Tff3 in diabetic/obese mice, presence in circulation and regulation by food/insulin, Tff3 is an interesting novel candidate in metabolism relevant conditions

Role of Sirt3 in Differential Sex-Related Responses to a High-Fat Diet in Mice

Metabolic homeostasis is differently regulated in males and females. Little is known about the mitochondrial Sirtuin 3 (Sirt3) protein in the context of sex-related differences in the development of metabolic dysregulation. To test our hypothesis that the role of Sirt3 in response to a high-fat diet (HFD) is sex-related, we measured metabolic, antioxidative, and mitochondrial parameters in the liver of Sirt3 wild-type (WT) and knockout (KO) mice of both sexes fed with a standard or HFD for ten weeks. We found that the combined effect of Sirt3 and an HFD was evident in more parameters in males (lipid content, glucose uptake, pparγ, cyp2e1, cyp4a14, Nrf2, MnSOD activity) than in females (protein damage and mitochondrial respiration), pointing towards a higher reliance of males on the effect of Sirt3 against HFD-induced metabolic dysregulation. The male-specific effects of an HFD also include reduced Sirt3 expression in WT and alleviated lipid accumulation and reduced glucose uptake in KO mice. In females, with a generally higher expression of genes involved in lipid homeostasis, either the HFD or Sirt3 depletion compromised mitochondrial respiration and increased protein oxidative damage. This work presents new insights into sex-related differences in the various physiological parameters with respect to nutritive excess and Sirt3

Chronic high fat diet intake impairs hepatic metabolic parameters in ovariectomized Sirt3 KO mice

High fat diet (HFD) is an important factor in the development of metabolic diseases, with liver as metabolic center being highly exposed to its influence. However, the effect of HFD-induced metabolic stress with respect to ovary hormone depletion and sirtuin 3 (Sirt3) is not clear. Here we investigated the effect of Sirt3 in liver of ovariectomized and sham female mice upon 10 weeks of feeding with standard-fat diet (SFD) or HFD. Liver was examined by Folch, gas chromatography and lipid hydroperoxide analysis, histology and oil red staining, RT-PCR, Western blot, antioxidative enzyme and oxygen consumption analyses. In SFD-fed WT mice, ovariectomy increased Sirt3 and fatty acids synthesis, maintained mitochondrial function, and decreased levels of lipid hydroperoxides. Combination of ovariectomy and Sirt3 depletion reduced pparα, Scd-1 ratio, MUFA proportions, CII-driven respiration, and increased lipid damage. HFD compromised CII-driven respiration and activated peroxisomal ROS scavenging enzyme catalase in sham mice, whereas in combination with ovariectomy and Sirt3 depletion, increased body weight gain, expression of NAFLD- and oxidative stress-inducing genes, and impaired response of antioxidative system. Overall, this study provides evidence that protection against harmful effects of HFD in female mice is attributed to the combined effect of female sex hormones and Sirt3, thus contributing to preclinical research on possible sex-related therapeutic agents for metabolic syndrome and associated diseases