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Organisms store fatty acids in triacylglycerols in the form of lipid droplets, or hydrolyze triacylglycerols in response to energetic demands via activation of lipolytic or storage pathways. These pathways are complex sets of sequential reactions that are finely regulated in different cell types. Here we present a high spatial and temporal resolution-based method for the quantification of the turnover of fatty acids into triglycerides in live cells without introducing sample preparation artifacts. We performed confocal spectral imaging of intracellular micropolarity in cultured insulin secreting beta cells to detect micropolarity variations as they occur in time and at different pixels of microscope images. Acquired data are then analyzed in the framework of the spectral phasors technique. The method furnishes a metabolic parameter, which quantitatively assesses fatty acids - triacylglycerols turnover and the activation of lipolysis and storage pathways. Moreover, it provides a polarity profile, which represents the contribution of hyperpolar, polar and non-polar classes of lipids. These three different classes can be visualized on the image at a submicrometer resolution, revealing the spatial localization of lipids in cells under physiological and pathological settings. This new method allows for a fine-tuned, real-time visualization of the turnover of fatty acids into triglycerides in live cells with submicrometric resolution. It also detects imbalances between lipid storage and usage, which may lead to metabolic disorders within living cells and organisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Lipids are molecules with different structures which have the feature of water insolubility in common. They have very important biological roles within structural, functional and signaling activities that have recently received renewed attention from life science research. Lipidomics considers the structural and functional roles played by lipids, but also their in vivo changes due to metabolic or degradation pathways, as well as their biological consequences. In this context, the dynamic vision of phospholipid metabolism and, in particular, fatty acid transformations combine with nutritional aspects and health consequences, providing important information for molecular medicine. Fatty acid-based functional lipidomics can be successfully applied to the follow-up of human lipid profiles under normal and pathological conditions, and this review provides several examples of this powerful molecular diagnostic tool, which is expected to have a strong influence on biomedical research in the 21st century.

Damage to bovine pancreatic RNase A, due to the H* atom and/or solvated electron attack at protein sulfur-containing residues, was investigated by Raman spectroscopy and mass spectrometry techniques. To the already known desulfurization process affecting Met residues, novel reactivity was observed involving disulfide moieties, leading to the chemical transformation of Cys into Ala residues. Mapping experiments demonstrated that desulfurization selectively affected Met79, Cys110, Cys58 and Cys72 during first stages of reaction. While this reaction was performed on protein species added to large unilamellar vescicles, desulfurization yielded sulfur radicals able to induce a cis-trans isomerization of lipids at the onset of irradiation. These findings reveal new scenarios on reactions generated by radical stressing conditions, suggesting the need for specific assays and for future investigations to detect these modifications in proteins and lipids within challenged cells.

The ability to survive to an extremely old age is a consequence of complex interactions among genes, environment, lifestyle and luck. In the last two centuries, life expectancy in western countries has doubled, increasing from 40 to 81 years (79 for males and 82 for females). The candidate factors to determine such mortality reduction are reduced exposure to infections and the subsequent reduction in inflammatory responses, and to some extent, improvement in diet and nutrition. Among the people born at the beginning of the previous century, a small portion of individuals (1 in 10,000 born) have reached 100 years, surviving approximately 20 years more than the general population. The successful longevity of these individuals shows a familial component, possibly genetic, as underlined by the centenarian sibling's increased chance of reaching 100 years of age compared to the general population. Genetic studies on long living individuals have led to the discovery of potential genetic causes of extreme longevity. These discoveries have highlighted the role of lipid metabolism as a potential key player in the ability to survive to extreme old age. Additional studies on the longevity phenotype have confirmed the role of lipids and lipid-associated cell activities in the predisposition to longevity, from lower eukaryotes to humans. The main focus of this review is the appreciation of demographic survival data and changes in recent diet with the above mentioned genetic and phenotypic biomarkers of longevity, in order to elucidate hypotheses on mechanisms of slow aging and disease resistance.

The occurrence of tandem damage, due to reductive radical stress involving proteins and lipids, is shown by using a biomimetic model. It is made of unsaturated lipid vesicle suspensions in phosphate buffer in the presence of methionine, either as a single amino acid or as part of a protein such as RNase A, which contains four methionine residues. The radical process starts with the formation of H(.) atoms by reaction of solvated electrons with dihydrogen phosphate anions, which selectively attack the thioether function of methionine. The modification of methionine to alpha-aminobutyric acid is accompanied by the formation of thiyl radicals, which in turn cause the isomerization of the cis fatty acid residues to the trans isomers. The relationship between methionine modification and lipid damage and some details of the reductive radical stress obtained by proteomic analysis of irradiated RNase A are presented.

Free radical-catalyzed cis-trans isomerization of unsaturated lipids and its outcome in biomimetic conditions are reviewed. They provided indications for an endogenous origin of trans unsaturated fatty acids in humans by the attack of thiyl radicals. A multidisciplinary approach highlights the relevance of this topic. The role of cis lipid geometry and its maintenance in the biological environment is an emerging question, with future developments in the fields of lipidomics, biology, and medicine.

Lipid membrane turnover and myelin repair play a central role in diseases and lesions of the central nervous system (CNS). The aim of the present study was to analyze lipid composition changes due to inflammatory conditions. We measured the fatty acid (FA) composition in erythrocytes (RBCs) and spinal cord tissue (gas chromatography) derived from mice affected by experimental allergic encephalomyelitis (EAE) in acute and remission phases; cholesterol membrane content (Filipin) and GM1 membrane assembly (CT-B) in EAE mouse RBCs, and in cultured neurons, oligodendroglial cells and macrophages exposed to inflammatory challenges. During the EAE acute phase, the RBC membrane showed a reduction in polyunsaturated FAs (PUFAs) and an increase in saturated FAs (SFAs) and the omega-6/omega-3 ratios, followed by a restoration to control levels in the remission phase in parallel with an increase in monounsaturated fatty acid residues. A decrease in PUFAs was also shown in the spinal cord. CT-B staining decreased and Filipin staining increased in RBCs during acute EAE, as well as in cultured macrophages, neurons and oligodendrocyte precursor cells exposed to inflammatory challenges. This regulation in lipid content suggests an increased cell membrane rigidity during the inflammatory phase of EAE and supports the investigation of peripheral cell membrane lipids as possible biomarkers for CNS lipid membrane concentration and assembly. [ABSTRACT FROM AUTHOR]

Gestational diabetes mellitus (GDM) is a glucose intolerance that begins or is first recognized during pregnancy. It is currently a growing health problem worldwide affecting from 1% to 14% of all pregnant women depending on racial and ethnic group as well as the diagnostic and screening criteria. Our preliminary study aimed at investigating the erythrocyte membrane fatty acid profiles of pregnant women, in particular with diagnosed with gestational diabetes mellitus (GDM), and with normal glucose tolerant (NGT) pregnant women as a control group. The study group comprised 43 pregnant women, 32 of whom were diagnosed with GDM according to the WHO criteria, and 11 with normal glucose tolerance. The erythrocyte membrane phospholipids were obtained according to the Folch extraction procedure. Fatty acids (FA) were analyzed by gas chromatography (GC) as the corresponding fatty acid methyl esters (FAME). A cluster of 14 fatty acids identified contained >98% of the recognized peaks in the GC analysis. The analysis of fatty acids from erythrocytes revealed important differences between GDM and NGT women in the third trimester, and the results were correlated with biochemical data. Among the 14 measured FA representing the membrane lipidomic profile, the levels of three saturated FA (myristic, palmitic, stearic acids) tended to decrease in GDM patients, with the percentage content of stearic acid significantly changed. The relative content of monounsaturated fatty acids (MUFA) tended to increase, in particular the oleic acid and vaccenic acid contents were significantly increased in erythrocyte membranes of the GDM group in comparison with the NGT group. The GDM group demonstrated higher sapienic acid levels (+29%) but this change was not statistically significant. This study revealed association between an impaired cis-vaccenic acid concentration in erythrocytes membrane and GDM development. No significant changes of polyunsaturated fatty acids (PUFA) were observed in GDM and NGT erythrocytes. We postulate, basing on the differences between the GDM and NGT lipidomic profiles, that stearic and cis-vaccenic acids can be considered as dual biomarkers of specific SFA-MUFA conversion pathway, involving the coupling of delta-9 desaturase and elongase enzymes. Our results indicate that the SFA-MUFA families may be involved in the pathophysiology of metabolic diseases such as GDM, but the further studies are needed to confirm our hypothesis. In conclusion, the erythrocyte membranes of GDM women undergo remodeling resulting in abnormal fatty acid profiles, which are reflection of the long-term status of organism and can have great impact on both the mother and her offspring. [ABSTRACT FROM AUTHOR]

Cis lipids can be converted by thiols and free radicals into trans lipids, which are therefore a valuable tell-tale for free radical activity in the cell’s lipidome. Our previous studies have shown that polyunsaturated lipids are isomerized by alkanethiyl radicals (S•) in a cycle propagated by reversible double-bond addition and terminated by radical H-abstraction from the lipid. A critical flaw in this picture has long been that the reported lipid abstraction rate from radiolysis studies is faster than addition–isomerization, implying that the “cycle” must be terminating faster than it is propagating! Herein, we resolved this longstanding puzzle by combining a detailed product analysis, with reinvestigation of the time-resolved kinetics, DFT calculations of the indicated pathways, and reformulation of the radical-stasis equations. We have determined thiol-coupled products in dilute solutions arise mainly from addition to the inside position of the bisallylic group, followed by rapid intramolecular H• transfer, yielding allylic radicals (LZZ + S• ⇄ SL• → SL′•) that are slowly reduced by thiol (SL′• + SH → SL′H + S•). The first-order grow-in rate of the L–H• signal (kexp280nm) may therefore be dominated by the addition-H-translocation rather than slower direct H•-abstraction. Steady-state kinetic analysis of the new mechanism is consistent with products and the rates and trends for polyunsaturated fatty acids (PUFAs), monounsaturated fatty acids (MUFAs), and mixtures, with and without physiological [O2]. Implications of this new paradigm for the thiol-ene reactivity fall in an interdisciplinary research area spanning from synthetic applications to metabolomics. [ABSTRACT FROM AUTHOR]

The prevalence of the metabolic syndrome components including abdominal obesity, dyslipidaemia and insulin resistance is increasing in both developed and developing countries. It is generally accepted that the development of these features is preceded by, or accompanied with, impaired mitochondrial function. The present study was designed to analyse the effects of a mitochondrial-targeted lipophilic ubiquinone (MitoQ) on muscle lipid profile modulation and mitochondrial function in obesogenic diet-fed rats. For this purpose, twenty-four young male Sprague–Dawley rats were divided into three groups and fed one of the following diets: (1) control, (2) high fat (HF) and (3) HF+MitoQ. After 8 weeks, mitochondrial function markers and lipid metabolism/profile modifications in skeletal muscle were measured. The HF diet was effective at inducing the major features of the metabolic syndrome – namely, obesity, hepatic enlargement and glucose intolerance. MitoQ intake prevented the increase in rat body weight, attenuated the increase in adipose tissue and liver weights and partially reversed glucose intolerance. At the muscle level, the HF diet induced moderate TAG accumulation associated with important modifications in the muscle phospholipid classes and in the fatty acid composition of total muscle lipid. These lipid modifications were accompanied with decrease in mitochondrial respiration. MitoQ intake corrected the lipid alterations and restored mitochondrial respiration. These results indicate that MitoQ protected obesogenic diet-fed rats from some features of the metabolic syndrome through its effects on muscle lipid metabolism and mitochondrial activity. These findings suggest that MitoQ is a promising candidate for future human trials in the metabolic syndrome prevention. [ABSTRACT FROM PUBLISHER]

Monounsaturated fatty acids (MUFA) are emerging health biomarkers, and in particular the ratio between palmitoleic acid (9cis-16:1) and palmitic acid (16:0) affords the delta-9 desaturase index that is increased in obesity. Recently, other positional and geometrical MUFA isomers belonging to the hexadecenoic family (C16 MUFA) were found in circulating lipids, such as sapienic acid (6cis-16:1), palmitelaidic acid (9trans-16:1) and 6trans-16:1. In this work we report: i) the identification of sapienic acid as component of human erythrocyte membrane phospholipids with significant increase in morbidly obese patients (n = 50) compared with age-matched lean controls (n = 50); and ii) the first comparison of erythrocyte membrane phospholipids (PL) and plasma cholesteryl esters (CE) in morbidly obese patients highlighting that some of their fatty acid levels have opposite trends: increases of both palmitic and sapienic acids with the decrease of linoleic acid (9cis,12cis-18:2, omega-6) in red blood cell (RBC) membrane PL were reversed in plasma CE, whereas the increase of palmitoleic acid was similar in both lipid species. Consequentially, desaturase enzymatic indexes gave different results, depending on the lipid class used for the fatty acid content. The fatty acid profile of morbidly obese subjects also showed significant increases of stearic acid (C18:0) and C20 omega-6, as well as decreases of oleic acid (9cis-18:1) and docosahexaenoic acid (C22:6 omega-3) as compared with lean healthy controls. Trans monounsaturated and polyunsaturated fatty acids were also measured and found significantly increased in both lipid classes of morbidly obese subjects. These results highlight the C16 MUFA isomers as emerging metabolic marker provided that the assignment of the double bond position and geometry is correctly performed, thus identifying the corresponding lipidomic pathway. Since RBC membrane PL and plasma CE have different fatty acid trends, caution must also be used in the choice of lipid species for the interpretation of lipidomic profiles. [ABSTRACT FROM AUTHOR]

Palmitic acid is known to be apoptotic for nervous cells but no data are available on membrane lipidome transformations occurring during its supplementation, although membrane lipids are clearly involved in the apoptotic signaling cascade. NB100 neuroblastoma cells were supplemented with palmitic acid and membrane fatty acids were isolated, derivatized and analysed by gas chromatography at defined time intervals. Parallely, cell viability, morphology, apoptosis, cPLA2 and caspase activations were checked. Interestingly, under 150 µM supplementation the incorporation of palmitic acid was accompanied by the specific release of arachidonic acid. This event was timely correlated with cPLA2 and caspases activations, and the time window of 60 minutes was envisaged for crucial membrane lipidome changes. The simultaneous addition of 50 µM oleic, 50 µM arachidonic and 150 µM palmitic acids to the cell cultures influenced membrane changes with suppression of caspase activation and maintenance of cell viability. These results highlight the role of the membrane asset with fatty acid remodeling and suggest the potential of lipid-based strategies for influencing cell response and fate in human diseases, such as neurodegenerative disorders or tumours. [ABSTRACT FROM AUTHOR]

Dietary lipids are known to affect the composition of the biological membrane and functions that are involved in cell death and survival. The mitochondrial respiratory chain enzymes are membrane protein complexes whose function depends on the composition and fluidity of the mitochondrial membrane lipid. The present study aimed at investigating the impact of different nutritional patterns of dietary lipids on liver mitochondrial functions. A total of forty-eight Wistar male rats were divided into six groups and fed for 12 weeks with a basal diet, lard diet or fish oil diet, containing either 50 or 300 g lipid/kg. The 30 % lipid intake increased liver NEFA, TAG and cholesterol levels, increased mitochondrial NEFA and TAG, and decreased phospholipid (PL) levels. SFA, PUFA and unsaturation index (UI) increased, whereas MUFA and trans-fatty acids (FA) decreased in the mitochondrial membrane PL in 30 % fat diet-fed rats compared with 5 % lipid diet-fed rats. PL UI increased with fish oil diet v. basal and lard-rich diets, and PL trans-FA increased with lard diet v. basal and fish oil diets. The 30 % lipid diet intake increased mitochondrial membrane potential, membrane fluidity, mitochondrial respiration and complex V activity, and decreased complex III and IV activities. With regard to lipid quality effects, β-oxidation decreased with the intake of basal or fish oil diets compared with that of the lard diet. The intake of a fish oil diet decreased complex III and IV activities compared with both the basal and lard diets. In conclusion, the characteristics and mitochondrial functions of the rat liver mitochondrial membrane are more profoundly altered by the quantity of dietary lipid than by its quality, which may have profound impacts on the pathogenesis and development of non-alcoholic fatty liver disease. [ABSTRACT FROM PUBLISHER]

Abstract: Trans lipids in humans originate exogenously from the ingestion of isomerized fats. An endogenous path comprising a thiyl radical-catalyzed cis–trans isomerization of cis-unsaturated phospholipids was proposed. However, whether an isomerization process might be feasible in eukaryotic cells remained to be established. Here we report the presence of trans lipids in human monocytic leukemia cell membranes (THP-1) before and after treatment with a 10 mM series of thiols. Oleic, linoleic, and arachidonic acid residues of membrane phospholipids were analyzed and, unexpectedly, an initial trans lipid content was found in control cells. Then, incubation for 24 h with thiols under physiological conditions slightly increased trans lipid content. Formation of trans isomers was also evaluated in the presence of thiol and under free radical stress induced by γ-irradiation or by thermal decomposition of azo-compounds. The similarity of isomer trends formed under incubation and stress conditions, together with the reactivity order of fatty acid residues (arachidonic > linoleic ≈ oleic), indicated a common radical path and some mechanistic considerations are advanced. These results offer the first evidence that trans lipids are formed in eukaryotic cells and confirm that thiyl radicals are harmful to the integrity of cis lipid geometry. This work motivates further studies into the relationship between lipid isomerization outcome and thiyl radicals in cellular systems, as well as the formation of trans lipids and the metabolic response to such a perturbation introduced into biological membranes. [Copyright &y& Elsevier]

Trans unsaturated fatty acids in humans may be originated by two different contributions. The exogenous track is due to dietary supplementation of trans fats and the endogenous path deals with free- radical-catalyzed cis--trans isomerization of fatty acids. Arachidonic acid residue (5c,8c,1lc,14c-20:4), which has only two out of the four double bonds deriving from the diet, was used to differentiate the two paths and to assess the importance of a radical reaction. A detailed study on the formation of trans phospholipids catalyzed by the HOCH[SUB2]CH[SUB2]S[SUP˙] radical was carried out on L-α-phosphatidylcholine from egg lecithin and 1 -stearoyl-2-arachidonoyl-L-α-phosphatidylcholine (SAPC) in homogeneous solution or in large unilamellar vesicles (LUVET). Thiyl radicals were generated from the corresponding thiol by either γ-irradiation or UV photolysis, and the reaction course was followed by GC, Ag/TLC, and [SUP13]C NMR analyses. The isomerization was found to be independent of cis double bond location (random process) in i-PrOH solution. In the case of vesicles, the supramolecular organization of lipids produced a dramatic change of the isomerization outcome: (i) in egg lecithin, the reactivity of arachidonate moieties is higher than that of oleate and linoleate residues, (ii) in the linoleate residues of egg lecithin, the 9t,12c-18:2 isomer prevailed on the 9c,12t-18:2 isomer (3:1 ratio), and (iii) a regioselective isomerization of SAPC arachidonate residues occurred in the 5 and 8 positions. This effect of "positional preference" indicates that thiyl radicals entering the hydrophobic region of the membrane bilayer start to isomerize polyunsaturated fatty acid residues having the double bonds nearest to the membrane surfaces. We propose that arachidonic acid and its trans isomers can function as biomarkers in membranes for distinguishing the two trans fatty acid-forming pathways. [ABSTRACT FROM AUTHOR]

The presence of trans fatty acids in mammalians is attributed to exogenous sources; nevertheless, trans isomers could be easily formed by free radical-catalyzed isomerization processes in vivo. The isomerization of methyl arachidonate (all-cis isomer) catalyzed by thiyl radical is proposed as a methodology applicable in biochemical laboratories, which produces mono- and di-trans isomers. Carbon-13 nuclear magnetic resonance spectroscopy shows that the carbon atom in position 15 is characteristic for each mono- and di-trans isomer. Antioxidants, such as α-tocopherol and all-trans-retinol acetate, inhibited the isomerization process. Trans phospholipids are formed in erythrocyte membranes by exposing blood to γ-irradiation in the presence of thiols, which is in contradiction with the known role of these compounds as radioprotectors. Trans isomers are also analyzed in tissues harvested from breast cancer patients and compared to the adipose breast tissue taken a few centimeters from the edge of the tumor from the same patient. This work is generally aimed at contributing to the debate on trans fatty acids and stimulating a reconsideration of the current view on the exclusive presence of cis double bonds in cell membranes by studying radical processes that could affect or protect this natural configuration. [Copyright &y& Elsevier]

A new pathway leading to the n-10 fatty acid series has been recently evidenced, starting from sapienic acid, a monounsaturated fatty acid (MUFA) resulting from the transformation of palmitic acid by delta-6 desaturase. Sapienic acid has attracted attention as a novel marker of cancer cell plasticity. Here, we analyzed fatty acids, including the n-10 fatty acid contents, and for the first time, compared cell membranes and the corresponding extracellular vesicles (EV) of two human prostatic adenocarcinoma cell lines of different aggressiveness (PC3 and LNCaP). The n-10 components were 9–13% of the total fatty acids in both cancer cell lines and EVs, with total MUFA levels significantly higher in EVs of the most aggressive cell type (PC3). High sapienic/palmitoleic ratios indicated the preference for delta-6 versus delta-9 desaturase enzymatic activity in these cell lines. The expressions analysis of enzymes involved in desaturation and elongation by qRT-PCR showed a higher desaturase activity in PC3 and a higher elongase activity toward polyunsaturated fatty acids than toward saturated fatty acids, compared to LNCaP cells. Our results improve the present knowledge in cancer fatty acid metabolism and lipid phenotypes, highlighting EV lipidomics to monitor positional fatty acid isomer profiles and MUFA levels in cancer. [ABSTRACT FROM AUTHOR]

The natural peptide somatostatin has hormonal and cytostatic effects exerted by the binding to specific receptors in various tissues. Therapeutic uses are strongly prevented by its very short biological half-life of 1–2 min due to enzymatic hydrolysis, therefore encapsulation methodologies are explored to overcome the need for continuous infusion regimes. Multilamellar liposomes made of natural phosphatidylcholine were used for the incorporation of a mixture of somatostatin and sorbitol dissolved in citrate buffer at pH = 5. Lyophilization and reconstitution of the suspension were carried out, showing the flexibility of this preparation. Full characterization of this suspension was obtained as particle size, encapsulation efficiency and retarded release properties in aqueous medium and human plasma. Liposomal somatostatin incubated at 37 °C in the presence of Fe(II) and (III) salts were used as a biomimetic model of drug-cell membrane interaction, evidencing the free radical processes of peroxidation and isomerization that transform the unsaturated fatty acid moieties of the lipid vesicles. This study offers new insights into a liposomal delivery system and highlights molecular reactivity of sulfur-containing drugs with its carrier or biological membranes for pharmacological applications. [ABSTRACT FROM AUTHOR]

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 thecis–transisomerization. Under anaerobic conditions,cis–transisomerization 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. [ABSTRACT FROM AUTHOR]

Review: 25 refs. [ABSTRACT FROM AUTHOR]

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 α-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 α-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. [Copyright &y& Elsevier]