Membrane Lipid Profile Research Articles

Maturation of buffalo oocytes in vitro with acetyl-L-carnitine improves cryotolerance due to changes in mitochondrial function and the membrane lipid profile.

The effects of acetyl-l-carnitine (ALC) supplementation during IVM on subsequently vitrified buffalo oocytes were evaluated, followed by determination of the mitochondrial DNA copy number, measurement of mitochondrial membrane potential (MMP) and identification of the lipid profile of oocyte membranes as markers of oocyte quality after vitrification. Supplementation with ALC during IVM significantly improved the rates of oocyte cleavage and morula and blastocyst formation, and increased MMP after vitrification compared with unsupplemented vitrified oocytes (P<0.05). Using a bidirectional orthogonal projection to latent structures discriminant analysis based on positive ion matrix-assisted laser desorption ionisation time-of-flight mass spectrometry data, five phospholipid ions (m/z 728.7 (phosphatidylcholine (PC) 32:3), 746.9 (PC 32:5), 760.6 (PC 34:1), 768.8 (PC P-36:3) and 782.6 (PC 36:4); P<0.05) were identified as significantly more abundant in fresh oocytes than in unsupplemented vitrified oocytes. Meanwhile, three phospholipid ions (m/z 734.6 (PC 32:0), 760.6 (PC 34:1), and 782.6 (PC 36:4); P<0.05) were more abundant in ALC-supplemented vitrified oocytes than in unsupplemented vitrified oocytes. Therefore, supplementation with ALC during IVM may improve buffalo oocyte quality after vitrification by enhancing mitochondrial function and altering the phospholipid composition of vitrified oocyte membranes.

NMR-Based Lipidomic Analysis of Red Blood Cells Membranes in Type 2 Diabetes

Changes in the composition of red blood cells membranes may lead to in an altered blood rheological pattern. Experimental studies suggest that hyperglycemia affects red cell rheology via direct effects on the membrane, including alterations of the lipid membrane bilayer composition and microviscosity and changes in membrane Na+/K+ ATP-ase function. We aimed to investigatate the 1H NMR-based lipid profile of RBC membranes in type 2 diabetic patients and compare it to that of nondiabetic subjects. Blood samples from 25 nondiabetic and 15 diabetic individuals were collected after an overnight fast. The groups were matched for age, sex and conventional lipid parameters. RBC were lysed and their lipid content was extracted according to a standard procedure. The lipid profile of membranes was recorded on Bruker DRX-500 NMR Spectrometer and analyzed by multivariate data techniques. The NMR-based lipidomic analysis showed that diabetic patients presented a significantly different lipid profile in RBC membranes compared to that recorded in nondiabetic individuals. RBC membranes from patients with diabetes were characterized by altered fatty acid pattern e.g., higher saturated and lower unsaturated fatty acid levels and lower concentrations of omega-3 fatty acids, mainly docosahexaenoic acid. In addition, lower levels of phosphatidylcholine and phosphatidylethanolamine, and higher levels of sphingomyelin and cholesterol were observed in diabetic patients compared to nondiabetic individuals. In conclusion, the lipid composition of RBC membranes in the diabetic state is significantly altered. These disturbances may be involved in the pathogenesis of the microvascular complications of diabetes through the disruption of tissue delivery of oxygen. Additionally, the increased cholesterol content of red cell membranes that reach the lipid core of the atherosclerotic plaques through the vasa vasorum may lead to core expansion, decrease their stability and make them prone to rupture. Disclosure C. Kostara: None. E. Bairaktari: None. M. Elisaf: None. V. Tsimihodimos: None.

Loss of Carotenoids Impacts Membrane Protein and Lipid Distribution in Pantoea sp. YR343

Bacterial cell membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties to the membrane and allows it to respond to environmental conditions. Pantoea sp. YR343 is a plant-associated Gram-negative proteobacteria that produces carotenoids, including zeaxanthin. We recently reported that Pantoea sp. YR343 mutants lacking the crtB gene encoding phytoene synthase, which catalyzes the first step in carotenoid biosynthesis, failed to produce carotenoids and displayed enhanced sensitivity to reactive oxygen species. The crtB mutant also displayed unexpected defects in biofilm formation and root colonization compared to wildtype cells. Studies using in silico molecular simulations suggest that carotenoids can act as modulators of membrane biophysical properties, leading to the possibility that these phenotypes may result from defects in membrane organization. We hypothesize that carotenoids modulate membrane dynamics by influencing the composition and organization of membrane proteins and lipids. Fluorescence anisotropy measurements on vesicles generated from lipids extracted from either wildtype or crtB mutant cells revealed that the wildtype membrane vesicles were significantly more rigid than mutant membrane vesicles. In contrast, anisotropy measurements on whole cells indicated that wildtype cells were less rigid than crtB mutant cells. These observations were confirmed by atomic force microscopy. Mass spectrometric based analysis of membrane proteins and lipids revealed differences in membrane protein and lipid profiles between the mutant and wildtype cells. Among the different classes of proteins, signaling and transport protein classes were the most effected in the mutant. Similarly, unsaturated and cyclopropane fatty acids classes differed in both cell types. These data suggest that loss of carotenoids in cell membranes changes the abundance and distribution of membrane proteins and lipids which may be the result of, or help compensate for, changes in membrane biophysical properties.

Erythrocytes and Skeletal Muscle Unsaturated and Omega-6 Fatty Acids Are Positively Correlated after Caloric Restriction and Exercise

Background: Nutritional intervention studies with fatty acid (FA) supplements assess the efficacy of the intervention by measuring the changes in erythrocyte membrane lipid profiles reflected in tissue composition changes. The aim was to determine the effects of caloric restriction (CR) on erythrocytes lipid composition and to compare and correlate these changes with skeletal muscle acid profiles after CR. Methods: Erythrocytes were obtained from 11 healthy men before and after 4 weeks of 33% CR in post-exercise conditions; muscle biopsies were obtained from the same athletes after 4 weeks of 33% CR in post-exercise conditions. Samples were used for FA determination by chromatography. Results: CR significantly modified erythrocyte FAs composition. Skeletal muscle FA profile was significantly different from that for the erythrocytes. The erythrocyte FA profile was more saturated (52.1 ± 1.5% and 32.8 ± 0.9%, respectively) and less monounsaturated (21.0 ± 0.8% and 39.0 ± 2.0%, respectively) than the skeletal muscle FA profile and similarly polyunsaturated. Conclusions: CR modifies erythrocyte lipid composition, mainly omega-6 FAs. Erythrocyte monounsaturated, polyunsaturated and omega-6 FAs, but not the saturated and omega-3 FAs, were significantly positively correlated with skeletal muscle FAs. There is a discordance between saturated and omega-3 FAs from erythrocyte and from muscle, but monounsaturated, polyunsaturated and omega-6 fatty acids are positively correlated.

ВЛИЯНИЕ ГЛИЦИРРИЗИНОВОЙ КИСЛОТЫ НА СОСТОЯНИЕ МЕМБРАННЫХ СТРУКТУР МОНОНУКЛЕАРОВ У БОЛЬНЫХ ТУБЕРКУЛЕЗОМ ЛЕГКИХ НА ФОНЕ ПРОТИВОТУБЕРКУЛЕЗНОЙ ХИМИОТЕРАПИИ

The objective of the study : to assess the lipid profile of membranes of peripheral blood mononuclear cells in the pulmonary tuberculosis patients before and after the intensive phase of chemotherapy, including the effect of glycyrrhizic acid. Subjects and methods . 384 pulmonary tuberculosis patients of both genders in the age from 25 to 60 years old were enrolled into the study: 308 patients were treated by regimen I during the intensive phase of chemotherapy, and 76 had the intensive phase with regimen I and glycyrrhizic acid. The control group included 36 healthy volunteers at the age from 24 to 58 years old. The lipid profile of mononuclear cell membranes was evaluated in peripheral blood of the patients. Phospholipids were fractioned into classes using continuous-flow thin-layer chromatography. The following fractions were studied: total lysophospholipids, sphingomyelin, phosphatidylinositol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine. Results . Prior to the start of chemotherapy, the changes in the ratio of main classes of phospholipids of mononuclear cell membranes in peripheral blood was documented in pulmonary tuberculosis patients versus those healthy. Chemotherapy resulted in the accumulation of membrane disruptive lysophospholipids with simultaneous reduction of phosphatidylcholine level. Treating patients with glycyrrhizic acid allowed diminishing (p < 0.05) the negative effect of chemotherapy on lipid membranes of mononuclear cells and improving the ratio of main fractions of membrane phospholipids (p < 0.05). When glycyrrhizic acid was added to the chemotherapy regimen, the sputum conversion rate by the 2nd month of the intensive phase increased from 61 to 73% (p < 0.05).

Haemato-protective influence of dietary fenugreek (Trigonella foenum-graecum L.) seeds is potentiated by onion (Allium cepa L.) in streptozotocin-induced diabetic rats

We have recently reported the beneficial modulation of metabolic abnormalities and oxidative stress in diabetic rats by dietary fenugreek seeds and onion. This investigation evaluated the protective influence of dietary fenugreek seeds (100 g kg−1) and onion (30 g kg−1) on erythrocytes of streptozotocin-induced diabetic rats, through modulation of reduced haematological indices and antisickling potency. This study also evaluated the altered erythrocyte membrane lipid profile and beneficial countering of increased lipid peroxidation, osmotic fragility, along with reduced membrane fluidity and deformability, nitric oxide production and echinocyte formation. Dietary fenugreek seeds and onion appeared to counter the deformity and fragility of erythrocytes partially in diabetic rats by their antioxidant potential and hypocholesterolemic property. The antisickling potency of these spices was accomplished by a substantial decrease in echinocyte population and AGEs in diabetic rats. Further insight into the factors that might have reduced the fluidity of erythrocytes in diabetic rats revealed changes in the cholesterol: phospholipid ratio, fatty acid profile, and activities of membrane-bound enzymes. Dietary fenugreek seeds and onion offered a beneficial protective effect to the red blood cells, the effect being higher with fenugreek + onion. This is the first report on the hemato-protective influence of a nutraceutical food component in diabetic situation.

LIPID METABOLITE PLASMA LEVELS AS BIOMARKERS OF EARLY-ONSET ALZHEIMER DISEASE

In recent years, in addition to the post-mortem diagnosis established practice, the demand of peripheral biomarkers of Alzheimer's disease (AD) has become a major focus of research due to the low cost and facility of access. The lipids and related molecules comprise 60% of the nonaqueous portion of the brain and are major constituents of membrane of neuronal and glial cells. Lipids play an increasingly recognized role in the neuronal function and plasticity of the brain. The lipid composition of brain substantially influences perception, mood and behavior. Since lipid metabolism is altered differently in neuropsychiatric diseases. Alterations in the membrane lipid profile can allow discrimination between patients with AD and MCI, thus suggesting a specificity of the findings for these diseases. Plasma is a good matrix in the search for candidates for biomarkers because it is easy to obtain and interacts with all tissues, providing an integration between the central and peripheral nervous system. Determine changes in the composition of plasma phospholipids and other metabolites, such as acylcarnitines, may help to elucidate the pathophysiological mechanisms in brain and the search for candidate biomarkers. Our objective was to determine plasma levels of lipids of patients with AD, MCI and healthy controls and propose metabolites candidates of peripheral biomarker and cutoff values capable to differentiate each diagnosis. Plasma of 32 patients with AD, 20 MCI and 30 healthy controls were analyzed by mass spectrometry - Flow injection analysis, using AbsoluteIDQ p180® kit (Biocrates Life Sciences). Statistical analyzes were performed using a classification method - Classification And Regression Tree. A combination of three metabolites are able to differentiate the diagnostic groups: Dodecanedioylcarnitine (C12-DC), Dodecanoylcarnitine (C12) and Phosphatidylcholine aa C26:0 (PC aa C26:0). The sensitivities are 64% for healthy controls, 80% for MCI and 79.4% for AD, with accuracy of 74.7%. Our results suggest that alterations of some plasma phospholipids levels may be previous of AD clinical signals and these alterations can differentiating MCI patients from healthy controls. Since these alterations are already present in the early cognitive deficits, the levels of these phospholipids can be a potential biomarker for early onset AD.

A reconfigured Kennedy pathway which promotes efficient accumulation of medium-chain fatty acids in leaf oils.

SummaryMedium‐chain fatty acids (MCFA, C6‐14 fatty acids) are an ideal feedstock for biodiesel and broader oleochemicals. In recent decades, several studies have used transgenic engineering to produce MCFA in seeds oils, although these modifications result in unbalance membrane lipid profiles that impair oil yields and agronomic performance. Given the ability to engineer nonseed organs to produce oils, we have previously demonstrated that MCFA profiles can be produced in leaves, but this also results in unbalanced membrane lipid profiles and undesirable chlorosis and cell death. Here we demonstrate that the introduction of a diacylglycerol acyltransferase from oil palm, EgDGAT1, was necessary to channel nascent MCFA directly into leaf oils and therefore bypassing MCFA residing in membrane lipids. This pathway resulted in increased flux towards MCFA rich leaf oils, reduced MCFA in leaf membrane lipids and, crucially, the alleviation of chlorosis. Deep sequencing of African oil palm (Elaeis guineensis) and coconut palm (Cocos nucifera) generated candidate genes of interest, which were then tested for their ability to improve oil accumulation. Thioesterases were explored for the production of lauric acid (C12:0) and myristic (C14:0). The thioesterases from Umbellularia californica and Cinnamomum camphora produced a total of 52% C12:0 and 40% C14:0, respectively, in transient leaf assays. This study demonstrated that the introduction of a complete acyl‐CoA‐dependent pathway for the synthesis of MFCA‐rich oils avoided disturbing membrane homoeostasis and cell death phenotypes. This study outlines a transgenic strategy for the engineering of biomass crops with high levels of MCFA rich leaf oils.

Streptococcus mutans membrane lipid composition: Virulence factors and structural parameters

ObjectiveThe aim of the present work was to analyze whether the location of the dental biofilm was associated with shifts in the membrane fatty acid profile, and whether such shifts could affect certain virulence factors of strains of Streptococcus mutans. DesignAn experimental study was conducted to assess the behavior of S. mutans strains isolated from dental biofilm collected from sound and carious smooth and occlusal surfaces of the oral cavity of children. The lipid composition of the bacterial membrane, structural membrane parameters, acid survival, and ATPase activity were tested at pH 7 and 5. ResultsAt pH 5, an increase in both the unsaturated and the long chain fatty acids as well as in proton ATPase hydrolytic activity was observed in strains isolated from carious smooth surface biofilm but not in carious occlusal surface strains. The observed changes correlated with the studied structural parameters, and were found to be associated with membrane organization. The changes correlated with a decreased in ΔC (sn-1 and sn-2 acyl chain inequivalence), indicative of increased acyl chain interaction. ConclusionsThe obtained results suggest that the acidic environment (pH 5) of smooth surface caries affects membrane organization, inducing a shift in membrane lipid profile, which would likely induce better protein/lipid hydrophobic matching, resulting in increased ATPase activity and higher acid survival.

Membrane lipid profiles of coral responded to zinc oxide nanoparticle-induced perturbations on the cellular membrane

Zinc oxide nanoparticles (nZnOs) released from popular sunscreens used during marine recreation apparently endanger corals; however, the known biological effects are very limited. Membrane lipids constitute the basic structural element to create cell a dynamic structure according to the circumstance. Nano-specific effects have been shown to mechanically perturb the physical state of the lipid membrane, and the cells accommodating the actions of nZnOs can be involved in the alteration of the membrane lipid composition. To gain insight into the effects of nanoparticles on coral, glycerophosphocholine (GPC) profiling of the coral Seriatopora caliendrum exposed to nZnOs was performed in this study. Increasing lyso-GPCs, docosapentaenoic acid-possessing GPCs and docosahexaenoic acid-possessing GPCs and decreasing arachidonic acid-possessing GPCs were the predominant changes responded to nZnO exposure in the coral. A backfilling of polyunsaturated plasmanylcholines was observed in the coral exposed to nZnO levels over a threshold. These changes can be logically interpreted as an accommodation to nZnOs-induced mechanical disturbances in the cellular membrane based on the biophysical properties of the lipids. Moreover, the coral demonstrated a difference in the changes in lipid profiles between intra-colonial functionally differentiated polyps, indicating an initial membrane composition-dependent response. Based on the physicochemical properties and physiological functions of these changed lipids, some chronic biological effects can be incubated once the coral receives long-term exposure to nZnOs.

Abstract P4-21-29: HER2-associated lipogenic phenotype as a potential therapeutical target in breast cancer patients

Abstract Lipogenic phenotype (genetic program activation involved inde novo fatty acid –FA-synthesis and key metabolic regulators) is associated to oncogenic transformation and malignancy. We hypothesize that in HER2 overexpressing breast cancer cells, HER2 function is affected by cell membrane lipid profile, mainly lipid rafts, necessary to HER2 location and activation within the cell membrane. Therefore, metabolic lipid imbalance could be a potential factor and marker associated to HER2 expression. To evaluate if cell lipogenesis and membrane lipid composition are affected by HER2 overexpression, we used an oncogenic transformed cell line engineered to overexpress HER2 receptor (HB4aC5.2), but identical to their parental strain (HB4a) in all other aspects. Several lipid related molecules including synthesis-FASN, uptake-CD36, transport-FABP4 and FA storage-DGAT by RT-PCR; and lipogenic regulatory pathways (mTOR, DEPTOR, SREBP1 and PPARγ) were evaluated in both cells. Lipogenic contribution to lipid rafts formation was evaluated by gas chromatography and confocal microscopy. The influence of HER2 overexpression and lipogenic phenotype on proteins activated by HER2 (AKT, ERK1/2 and FASN) was analyzed by western blot. Next, HB4a and HB4aC5.2 cells were treated, alone or in combination with DHA (omega-3 polyunsaturated fatty acid), by Trastuzumab (anti-HER2), and GW9662 (anti-PPARγ) for 72h, and all above experiments were repeated. Cell death was analyzed by flow cytometry. In vitro results were confirmed in Primary tumor and adjacent tissue samples from a cohort of 182 European breast cancer patients through metabolomics analysis. Statistical analysis was performed by MetaboAnalyst software (p &amp;lt;0.05). In HB4aC5.2 cells, the HER2 overexpression was associated with a lipogenic phenotype, which increase in cholesterol and saturated FA synthesis for the lipid rafts formation, suggesting that large amounts of HER2 require cell membrane changes to favor rafts saturation and formation, when compared to HB4a. Lipid rafts were associated with survival and proliferation signals hiper activation (p&amp;lt;0.001). Cell lipogenesis was independent ofmTOR/SREBP/PPARγpathway and occur concomitantly to DEPTOR (mTOR inhibitor) increased expression. Moreover, GW9662 or Trastuzumab treatment did not influence lipogenesis in cell culture. In HB4aC5.2 cells, only DHA treatment decreased the lipogenic phenotype, DEPTOR expression, saturated FA synthesis, disrupted lipid raft, inhibited HER2 signaling, and induced apoptosis (p&amp;lt;0.001). In addition, when added to Trastuzumab DHA potentiated cell death. In tumor tissue samples an increased FA synthesis was observed, such as palmitic acid, sphingolipids and phosphatidylcholines saturated FA enriched, synthesis of cholesterol, suggesting an increase of lipid rafts when compared to normal breast adjacent tissue. Our results indicate that lipogenic phenotype is associated with HER2 superexpression in breast cancer cells, and it seems important to promote survival and proliferation, through lipid rafts increase. DHA modulation increased death in tumor cells, suggesting that cell membrane lipogenesis and lipid composition may represent a treatment target. Lipogenic phenotype confirmation in tumor tissue samples strengthens this hypothesis. Citation Format: Ravacci GR, Santos JR, Brentani MM, Tortelli T, Dale I, Logullo AF, Waitzberg DL. HER2-associated lipogenic phenotype as a potential therapeutical target in breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-21-29.

Membrane engineering of S. cerevisiae targeting sphingolipid metabolism

The sustainable production of fuels and chemicals using microbial cell factories is now well established. However, many microbial production processes are still limited in scale due to inhibition from compounds that are present in the feedstock or are produced during fermentation. Some of these inhibitors interfere with cellular membranes and change the physicochemical properties of the membranes. Another group of molecules is dependent on their permeation rate through the membrane for their inhibition. We have investigated the use of membrane engineering to counteract the negative effects of inhibitors on the microorganism with focus on modulating the abundance of complex sphingolipids in the cell membrane of Saccharomyces cerevisiae. Overexpression of ELO3, involved in fatty acid elongation, and AUR1, which catalyses the formation of complex sphingolipids, had no effect on the membrane lipid profile or on cellular physiology. Deletion of the genes ORM1 and ORM2, encoding negative regulators of sphingolipid biosynthesis, decreased cell viability and considerably reduced phosphatidylinositol and complex sphingolipids. Additionally, combining ELO3 and AUR1 overexpression with orm1/2Δ improved cell viability and increased fatty acyl chain length compared with only orm1/2Δ. These findings can be used to further study the sphingolipid metabolism, as well as giving guidance in membrane engineering.

Changes of membrane fatty acids and proteins of Shewanella putrefaciens treated with cinnamon oil and gamma irradiation

BackgroundIn order to detect the antimicrobial mechanism of combined treatment of cinnamon oil and gamma irradiation (GI), the membrane fatty acids and proteins characteristics of Shewanella putrefaciens (S. putrefaciens) treated with cinnamon oil and GI, and the distribution of cinnamon oil in S. putrefaciens were observed in this study.ResultsThe membrane lipid profile of S. putrefaciens was notably damaged by treatments of cinnamon oil and the combination of cinnamon oil and GI, with significantly fatty acids decrease in C14:0, C16:0, C16:1, C17:1, C18:1 (p < 0.05). The SDS-PAGE result showed that GI did not have obvious effect on membrane proteins (MP), but GI combined with cinnamon oil changed the MP subunits. Cinnamaldehyde, the main component of cinnamon oil, can not transport into S. putrefaciens obviously. It was transformed into cinnamyl alcohol in the nutrient broth with the action of S. putrefaciens. This indicated that the antimicrobial action of cinnamon oil mainly happened on the membrane of S. putrefaciens.ConclusionCinnamon oil could act on the membrane of S. putrefaciens with the damage of fatty acids and proteins, and GI would increase the destructive capability of cinnamon oil on the membrane fatty acids and proteins of S. putrefaciens.

Hormone deprivation alters mitochondrial function and lipid profile in the hippocampus

Mitochondrial dysfunction is a common hallmark in aging. In the female, reproductive senescence is characterized by loss of ovarian hormones, many of whose neuroprotective effects converge upon mitochondria. The functional integrity of mitochondria is dependent on membrane fatty acid and phospholipid composition, which are also affected during aging. The effect of long-term ovarian hormone deprivation upon mitochondrial function and its putative association with changes in mitochondrial membrane lipid profile in the hippocampus, an area primarily affected during aging and highly responsive to ovarian hormones, is unknown. To this aim, Wistar adult female rats were ovariectomized or sham-operated. Twelve weeks later, different parameters of mitochondrial function (O2 uptake, ATP production, membrane potential and respiratory complex activities) as well as membrane phospholipid content and composition were evaluated in hippocampal mitochondria. Chronic ovariectomy reduced mitochondrial O2 uptake and ATP production rates and induced membrane depolarization during active respiration without altering the activity of respiratory complexes. Mitochondrial membrane lipid profile showed no changes in cholesterol levels but higher levels of unsaturated fatty acids and a higher peroxidizability index in mitochondria from ovariectomized rats. Interestingly, ovariectomy also reduced cardiolipin content and altered cardiolipin fatty acid profile leading to a lower peroxidizability index. In conclusion, chronic ovarian hormone deprivation induces mitochondrial dysfunction and changes in the mitochondrial membrane lipid profile comparable to an aging phenotype. Our study provides insights into ovarian hormone loss-induced early lipidomic changes with bioenergetic deficits in the hippocampus that may contribute to the increased risk of Alzheimer's disease and other age-associated disorders observed in postmenopause.

Oral administration of fumonisin B1 and T-2 individually and in combination affects hepatic total and mitochondrial membrane lipid profile of rabbits.

Weaned rabbits were fed diets contaminated with 2mg/kg diet T-2 toxin alone, or 10mg/kg diet fumonisin B1 (FB1) alone, and both toxins in combination (2 + 10mg/kg, respectively) compared to a toxin-free control diet. Samplings were performed after 4 weeks (blood and liver). Bodyweight of T-2-fed group was lower after 4 weeks; the liver weight was increased dramatically (threefold of control). Liver total phospholipids (PLs) provided slight alterations in the fatty acid (FA) composition; all three toxin-treated groups showed a decrease in palmitoleic acid (C16:1 n7) proportion. In the liver mitochondrial PL FA composition, margaric acid (C17:0) proportion decreased in the separated toxin treatments compared to the combined setting. Oleic acid (C18:1 n9) proportion was increased and arachidonic acid (C20:4 n6) was decreased in the FB1-treated group, while docosapentaenoic acid (C22:5 n3) was decreased in the separated treatments. The total monounsaturation was significantly higher in the FB1 group's mitochondrial PL FA profile. After 4 weeks, all toxin treatments decreased the blood plasma reduced glutathione and glutathione peroxidase activity, and FB1 increased the plasma sphinganine/sphingosine ratio. Both mycotoxins seem to cross the hepatocellular and the hepatic mitochondrial membrane, without drastic membrane disruption, as assessed from the PL FA composition, but inducing detectable lipid peroxidation.

Plasma membrane lipid profile from resistant and sensitive spermatozoa of Mangalarga Marchador stallions after cooling at 5°C

Plasma membrane lipid profile from resistant and sensitive spermatozoa of Mangalarga Marchador stallions after cooling at 5°C

Effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine on membrane lipid profile and cryotolerance of human sperm

To study the effects of n-6 and n-3 polyunsaturated acid-rich soybean phosphatidylcholine (soy-PC) on sperm cryotolerance with regard to sperm membrane lipid profile, membrane surface integrity, and routine semen parameters. Experimental study. University-affiliated tertiary hospital. A total of 20 normospermic fertile men. Semen samples examined for differences in semen parameters, sperm membrane lipid profile, and plasma membrane surface both before and after cryopreservation using basic freezing medium with N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid (TES) and tris-(hydroxymethyl)-aminomethane (TRIS) supplemented with purified soy-PC (TEST-PC) or egg yolk (TEST-Y), both alone or in association (TEST-Y-PC). Conventional semen parameters and membrane lipid profile by matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS). Postthaw sperm cell motility, vitality, and morphology parameters were similar for soy-PC (TEST-PC) and egg yolk (TEST-Y) cryoprotectants. However, sperm exposed to TEST-Y-PC presented better kinetic parameters, which were similar to the original quality of the fresh semen. Human sperm MALDI-MS lipid profiles revealed that the relative abundance of glycerophospholipids of m/z 760.44 [PC (34:1)+H]+, 781.55 [SM (20:0) +Na]+, 784.55 [PC (36:3) +H]+, 806.64 [PC (38:6) +H]+, 807.64 [SM (22:1) +Na]+, and 809.64 [SM (22:0) +Na]+ increased in soy-PC samples (TEST-PC). Nonetheless, only one lipid (m/z 781.55, [SM (20:0) +Na]+) statistically significantly changed when sperm was cryopreserved in TEST-Y-PC. Sphingomyelin was defined as a prospective biomarker of soy-PC treatment, and it could be related to the positive cryoprotective effects of soy-PC in human sperm, opening new perspectives to design of a more efficient synthetic cryoprotectant medium containing purified egg yolk biomolecules combined with soy-PC.

Effects of bleomycin and antioxidants on the fatty acid profile of testicular cancer cell membranes

Bleomycin is used in chemotherapy regimens for the treatment of patients having testicular germ-cell tumor (TGCT). There is no study in the literature investigating the effects of bleomycin on membrane lipid profile in testicular cancer cells. We investigated membrane fatty acid (FA) profiles isolated, derivatized and analyzed by gas chromatography of NTera-2 testicular cancer cells incubated with bleomycin (Bleo) for 24h in the absence and presence of N-Acetyl-L-Cysteine (NAC) and curcumin (Cur) as commonly used antioxidant adjuvants. At the same time the MAPK pathway and EGFR levels were followed up. Bleomycin treatment increased significantly saturated fatty acids (SFA) of phospholipids at the expense of monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA). Bleomycin also led to a significant increase in the trans lipid isomers of oleic and arachidonic acids due to its free radical producing effect. Incubation with bleomycin increased the p38 MAPK and JNK levels and downregulated EGFR pathway. Coincubation of bleomycin with NAC reversed effects caused by bleomycin. Our results highlight the important role of membrane fatty acid remodeling occurring during the use of bleomycin and its concurrent use with antioxidants which can adjuvate the cytotoxic effects of the chemotherapeutic agents.

160 LIPIDOME SIGNATURES IN EARLY BOVINE EMBRYO DEVELOPMENT

Mammalian pre-implantation embryonic development is a complex, conserved, and well-orchestrated process involving dynamic molecular and structural changes. Understanding the membrane lipid profile fluctuation during this crucial period is fundamental to address cellular and molecular mechanisms governing embryogenesis. A full understanding of stage-specific lipid signatures in early bovine embryo development is, however, still lacking. The aim of the present work was to characterise stage-specific changes in lipid profiles during early bovine embryonic development. Immature oocytes were recovered from slaughterhouse-derived bovine ovaries and assigned among 5 in vitro developmental stages for lipid characterisation: immature oocytes, 2-cell embryos (32–40 h post-insemination), 8 to 16-cell embryos (72 h post-insemination), morulas (120 h post-insemination), and blastocysts (168 h post-insemination). Two different culture media were used for in vitro embryo production, SOFaaci medium supplemented with 2.5% of serum and serum-free SOF-BE1 medium. Cytoplasmic lipid droplets content and membrane phospholipids profiles for each development stage were assessed by lipid staining (Nile red; n = 5–9) and matrix-assisted laser desorption ionization as a mass spectrometry imaging (MALDI-MS; n = 5–9), respectively. For statistical analysis, univariate and multivariate models were used to compare lipid droplets content and membrane phospholipids profiles. Cytoplasmic lipid droplets content increased from minimum in the immature oocyte stage to maximum at the morula stage, followed by a sharp drop at the blastocyst stage (58.4 ± 10.5ac, 62.5 ± 9.4ac, 85.9 ± 8.2a, 148.3 ± 7.4b, 37.4 ± 9.9c of fluorescence intensity per embryo area, respectively, for immature oocyte, 2-cells, 8 to 16-cells, morulas, and blastocysts; abcP &lt; 0.05). More cytoplasmic lipid droplets were detected in morulas and blastocyts cultured in SOFaaci than in SOF-BE1 (morulas, 162.6 ± 11.3 v. 137.1 ± 9.2 of fluorescence intensity per embryo area, respectively, P &lt; 0.05; blastocysts, 49.9 ± 9.9 v. 20.7 ± 9.9 of fluorescence intensity per embryo area, respectively, P &lt; 0.05). Characteristic dynamic changes of unsaturation level, acyl chain length and class composition (phosphatidylcholines, sphingomyelins, and phosphatidylethanolamines) of phospholipid profiles were observed during early embryo development. This study provides a comprehensive analysis, involving lipid staining and mass spectrometry evaluation, of stage-specific lipid signatures during bovine in vitro embryo development. These results may be useful for assessing the role of specific lipid species during important events of embryogenesis. Research was supported by CNPq, FAPESP, and FAPERGS.

Cross-GenerationaltransFat Consumption Favors Self-Administration of Amphetamine and Changes Molecular Expressions of BDNF, DAT, and D1/D2 Receptors in the Cortex and Hippocampus of Rats.

Amphetamine (AMPH) is an addictive psychostimulant drug whose use has been related to neurotoxicity. Experimentally, AMPH increases anxiety-like symptoms, showing addictive properties. In the last decades, the growing consumption of processed foods has provided an excess of saturated and trans fats in detriment of essential fatty acids, which may modify the lipid profile of brain membranes, thus modifying its permeability and dopaminergic neurotransmission. Here, we assessed the influence of brain incorporation of different fatty acids (FA) on AMPH self-administration. Three groups of young male rats were orally supplemented from weaning with a mixture of soybean oil (SO, rich in n-6 FA) and fish oil (FO, rich in n-3 FA), hydrogenated vegetable fat (HVF, rich in trans fatty acids--TFA), or water (control group). These animals were born from dams that were supplemented with the same fat from pregnancy to lactation. Anxiety-like symptoms and locomotor index were assessed in elevated plus maze and open-field (OF), respectively, while brain molecular expressions of dopaminergic receptors, dopamine transporter (DAT), and BDNF were determined in the cortex and hippocampus. HVF increased the frequency of AMPH self-administration and was associated with reinforcement and withdrawal signs as observed by increased anxiety-like symptoms. Contrarily, SO/FO decreased these parameters. Increased BDNF protein together with decreased DAT expression was observed in the hippocampus of HVF group. Based on these findings, our study points to a harmful influence of trans fats on drug addiction and craving symptoms, whose mechanism may be related to changes in the dopaminergic neurotransmission.