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Our study aimed to show a relationship between metabolic control, vitamin D status (25OHD), and arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio in children with type 1 diabetes (T1D). The secondary aim was to evaluate dietary intake and the presence of ketoacidosis (DKA) at the onset of T1D. Methods: A cohort of 40 children with T1D was recruited, mean age 9.7 years (7.1; 13), with onset of T1D in the last 5 years: some at onset (n: 20, group A) and others after 18.0 ± 5 months (n: 20; group B). Twenty healthy children were compared as control subjects (CS). Dietary intakes were assessed through a diary food frequency questionnaire. Moreover, dried blood spots were used to test AA/EPA ratio by gas chromatography. Results: T1D children had a lower percentage of sugar intake (p < 0.02) than CS. Furthermore, group B introduced a greater amount of AA with the diet (g/day; p < 0.05) than CS (p < 0.01) and group A (p < 0.01). Children with an AA/EPA ratio ≤ 22.5 (1st quartile) required a lower insulin demand and had higher 25OHD levels than those who were in the higher quartiles (p < 0.05). Subjects with DKA (9/40) had levels of 25OHD (p < 0.05) and C-peptide (p < 0.05) lower than those without DKA. Moreover, analyzing the food questionnaire in group A, subjects with DKA showed a lower intake of proteins, sugars, fiber (g/day; p< 0.05), vitamin D, EPA, and DHA (g/day; p < 0.01) compared to subjects without DKA. Non-linear associations between vitamin D intake (p < 0.0001; r2:0.580) and linear between EPA intake and C-peptide (p < 0.05; r: 0.375) were found in all subjects. Conclusions: The study shows a relationship between vitamin D status, AA/EPA ratio, and metabolic state, probably due to their inflammatory and immune mechanisms. A different bromatological composition of the diet could impact the severity of the onset.

Vitamin D and ω-3 fatty acids (ω-3) co-supplementation potentially improve type 1 diabetes (T1D) by attenuating autoimmunity and counteracting inflammation.This cohort study preliminary to a randomized control trial (RCT) is aimed to evaluate, in a series of T1D children assuming Mediterranean diet and taking cholecalciferol 1000U/day fromT1D onset, if ω-3 co-supplementation preserves the residual endogen insulin secretion (REIS).Therefore, 22 new onsets of 2017 received ω-3 [eicosapentenoic acid (EPA) plus docosahexaenoic acid (DHA), 60mg/kg/day], and were compared retrospectively vs. 37 previous onsets without ω-3 supplementation. HbA1c%, daily insulin demand (IU/Kg/day) and IDAA1c, a composite index (calculated as IU/Kg/dayx4 + HbA1c%) as surrogate of REIS, were evaluated at recruitment (T0) and 12 months later (T12). In the ω-3 supplemented group, dietary intakes were evaluated at T0 and T12. As outcome, decreased insulin demand (p

Ischemic-reperfusion (I/R) injury induced a remodeling of protein and lipid homeostasis, under oxidative stress and inflammatory status. Starvation occurring during I/R is a condition leading to autophagy activation, which allows abnormal material clearance or amino acid, or both, and fatty acid (FA) recycling essential for survival. This study investigated the lipid reshaping, peroxidation, and related-signaling pathways, in rat brain endothelial cells (RBE4) subjected to 3 h of oxygen and glucose deprivation (OGD) and restoration of standard condition (I/R in vitro model). Lipids and proteins were analyzed after 1 or 24 h of oxygen and nutrient restoration. Together with the oxidative stress and inflammatory status, I/R injury induced a reshaping of neutral lipids and biogenesis of lipid droplets (LD) with excessive lipid storage. The increase of LC3-II/LC3-I ratio, an autophagy marker, and LC3 co-localization with LD suggest the activation of lipophagy machinery to counteract the cell engulfment. Lipophagy leads to cholesterol ester (CE) hydrolysis, increasing free cholesterol (FC) secretion, which occurred by specific transporters or unconventional exocytosis pathways, or both. Here, we propose that an unconventional spreading of FC and other lipid metabolites may influence the neurovascular unit (NVU) cells, contributing to Blood brain barrier (BBB) alteration or adaptation, or both, to the cumulative effects of several transient ischemia.

Aims: Changes in lipids metabolism under ischemia-reperfusion (I/R) injury is implicated in oxidative stress and inflammatory response that lead to blood brain barrier (BBB) break-down. An increase in cell membranes cholesterol outcomes in the amyloid-β (Aβ) production during OGD/ogR [1]. The present study is aimed to monitor lipids fluctuations and peroxidation as well signaling pathway linked to those, in order to understand their contribute in BBB dysfunctions. Materials: Rat brain endothelial (RBE4) cells shows typical endothelial morphology retaining many characteristics; 5%CO2: 95%N2 gaseous mixtures (Sapio); hypoxia chamber (Billups- Rothenberg). Methods: RBE4 cells are subjected to oxygen and glucose deprivation (OGD). After medium replacement with a glucose-free balanced salt solution, cells are incubated for 3 hours in a gaseous mixture saturated chamber. The restoration solution (glucose and FBS) is administrated to cells (ogR) for one hour or 24 hours. Lipids analyses are performed by means high performance liquid chromatography (HPLC). Results: After OGD followed by 1 or 24 h ogR, the enhancement of inflammatory (COX-2) and oxidative stress (MDA) markers is detected. Phospholipids levels do not change, while neutral lipids metabolism is significant altered. Triglycerides (TG) levels increase accumulating in lipid droplets (LD), which move from nuclear to plasma membrane following a specific direction of actin filaments during ogR (1-24h). In parallel, cholesterol ester (CE), gathered in LD, decrease about 40% at ogR1h, returning to the control level at ogR24h. Activation of autophagy pathway is revealed by the increase of LC3-II/LC3-I ratio and the decrease in p62 levels. Finally, immunofluorescence experiments, show an increase in LD and LC3 co-localization during ogR. Discussion: I/R injury seems to induce a reshaping in triglycerides and in CE, sustaining the lipid droplets biogenesis up to an excessive lipid storage. During ogR the autophagy pathway is significant activated to counteract the cell engulfment (lipids and protein), as showed by LD and LC3 co-localization. The movement of LD to plasma membrane, suggest that conventional autophagy might be flanked by exocytosis pathways. Moreover, the increase of CE hydrolysis might play a role in Aβ stabilization along to a diminished Aβ lysosomal degradation [2]. Conclusions: I/R injury leads to the reshaping in neutral lipids that alters cellular membrane and cellular pathways, including Aβ stabilization and accumulation. Nevertheless, unconventional lipid exocytosis (TG and CE) might represent a type of cell communication to counteract the harmful effects of ischemic insult [3]

Vitamin D and omega 3 fatty acid (&omega, 3) co-supplementation potentially improves type 1 diabetes (T1D) by attenuating autoimmunity and counteracting inflammation. This cohort study, preliminary to a randomized control trial (RCT), is aimed at evaluating, in a series of T1D children assuming Mediterranean diet and an intake of cholecalciferol of 1000U/day from T1D onset, if &omega, 3 co-supplementation preserves the residual endogen insulin secretion (REIS). Therefore, the cohort of 22 &ldquo, new onsets&rdquo, of 2017 received &omega, 3 (eicosapentenoic acid (EPA) plus docosahexaenoic acid (DHA), 60 mg/kg/day), and were compared retrospectively vs. the 37 &ldquo, previous onsets&rdquo, without &omega, 3 supplementation. Glicosilated hemoglobin (HbA1c%), the daily insulin demand (IU/Kg/day) and IDAA1c, a composite index (calculated as IU/Kg/day &times, 4 + HbA1c%), as surrogates of REIS, were evaluated at recruitment (T0) and 12 months later (T12). In the &omega, 3 supplemented group, dietary intakes were evaluated at T0 and T12. As an outcome, a decreased insulin demand (p &lt, 0.01), particularly as pre-meal boluses (p &lt, 0.01), and IDAA1c (p &lt, 0.05), were found in the &omega, 3 supplemented group, while HbA1c% was not significantly different. Diet analysis in the &omega, 3 supplemented group, at T12 vs. T0, highlighted that the intake of arachidonic acid (AA) decreased (p &lt, 0.01). At T0, the AA intake was inversely correlated with HbA1c% (p &lt, 0.05, r, 0.411). In conclusion, the results suggest that vitamin D plus &omega, 3 co-supplementation as well as AA reduction in the Mediterranean diet display benefits for T1D children at onset and deserve further investigation.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a form of Neurodegeneration with Brain Iron Accumulation (NBIA) associated with mutations in the pantothenate kinase 2 gene (PANK2). The PANK2 catalyzes the first step of coenzyme A (CoA) biosynthesis, a pathway producing an essential cofactor that plays a key role in energy and lipid metabolism. The majority of PANK2 mutations reduces or abolishes the activity of the enzyme. In around 10% of cases with PKAN, the presence of deformed red blood cells with thorny protrusions in the circulation has been detected. Changes in membrane protein expression and assembly during erythropoiesis were previously explored in patients with PKAN. However, data on red blood cell membrane phospholipid organization are still missing in this disease. In this study, we performed lipidomic analysis on red blood cells from Italian patients affected by PKAN with a particular interest in membrane physico-chemical properties. We showed an increased number of small red blood cells together with membrane phospholipid alteration, particularly a significant increase in sphingomyelin (SM)/phosphatidylcholine (PC) and SM/phosphatidylethanolamine (PE) ratios, in subjects with PKAN. The membrane structural abnormalities were associated with membrane fluidity perturbation. These morphological and functional characteristics of red blood cells in patients with PKAN offer new possible tools in order to shed light on the pathogenesis of the disease and to possibly identify further biomarkers for clinical studies.

Epidemiologic and experimental studies suggest that dietary fatty acids influence the development and progression of breast cancer. However, no clear data are present in literature that could demonstrate how n - 3 PUFA can interfere with breast cancer growth. It is suggested that these fatty acids might change the structure of cell membrane, especially of lipid rafts. During this study we treated MCF-7 and MDA-MB-231 cells with AA, EPA, and DHA to assess if they are incorporated in lipid raft phospholipids and are able to change chemical and physical properties of these structures. Our data demonstrate that PUFA and their metabolites are inserted with different yield in cell membrane microdomains and are able to alter fatty acid composition without decreasing the total percentage of saturated fatty acids that characterize these structures. In particular in MDA-MB-231 cells, that displays the highest content of Chol and saturated fatty acids, we observed the lowest incorporation of DHA, probably for sterical reasons; nevertheless DHA was able to decrease Chol and SM content. Moreover, PUFA are incorporated in breast cancer lipid rafts with different specificity for the phospholipid moiety, in particular PUFA are incorporated in PI, PS, and PC phospholipids that may be relevant to the formation of PUFA metabolites (prostaglandins, prostacyclins, leukotrienes, resolvines, and protectines) of phospholipids deriving second messengers and signal transduction activation. The bio-physical changes after n - 3 PUFA incubation have also been highlighted by atomic force microscopy. In particular, for both cell lines the DHA treatment produced a decrease of the lipid rafts in the order of about 20-30 %. It is worth noticing that after DHA incorporation lipid rafts exhibit two different height ranges. In fact, some lipid rafts have a higher height of 6-6.5 nm. In conclusion n - 3 PUFA are able to modify lipid raft biochemical and biophysical features leading to decrease of breast cancer cell proliferation probably through different mechanisms related to acyl chain length and unsaturation. While EPA may contribute to cell apoptosis mainly through decrease of AA concentration in lipid raft phospholipids, DHA may change the biophysical properties of lipid rafts decreasing the content of cholesterol and probably the distribution of key proteins.

Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.

The wound repair function of mare's milk and colostrum was investigated. Mare's colostrum improved wound healing in vivo; thus fibroblast growth activation by mare's milk and colostrum was examined. As expected, colostrum was more effective than milk. To establish the biochemical nature of the bioactive molecules involved, colostrum was fractionated into whey, casein, and fat globules, and the efficacy of these fractions on fibroblast proliferation was studied. The fat globule fraction provided the strongest stimulation; its composition was studied and compared with the less-active milk fat globule fraction. The lipid pattern highlighted several differences between mare's colostrum and milk; in particular, total lipid, linoleic acid, linolenic acid, ganglioside, and glycolipid contents were higher in colostrum. A proteomic investigation revealed some differences between the protein composition of colostrum and milk fat globules. Adipophylin and lactadherin were significantly overexpressed in colostrum fat globules. The role of specific lipids on skin wound repair and that of the epidermal growth factor-like domain, embedded within the lactadherin molecule and probably released in conditions stimulating proteolysis, are discussed.

The association of dietary fats with disease risk or outcome can be determined from epidemiological studies and/or from food frequency questionnaires; a better assessment of the dietary intake of the long-chain polyunsaturated fatty acids omega-3 is obtained by the fatty acid composition of the platelets or of erythrocyte membranes yet these procedures are lengthy. Other investigators have used adipose tissue obtained by percutaneous biopsy, but it must be pointed out that this procedure is not only invasive but time-consuming as well. Also, it has to be noted that the turnover time for fatty acids has been estimated to be 1–3 years.

The ABCB4 gene encodes for MDR3, a protein that translocates phosphatidylcholine from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency favors the formation of ‘toxic bile'. Several forms of hepatobiliary diseases have been associated with ABCB4 mutations, but the detrimental effects of most mutations on the encoded protein needs to be clarified. Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. This study was aimed at analyzing the functional relevance of these two ABCB4 mutations: MDR3 expression and lipid content in the culture supernatant were evaluated in cell lines stably transfected with the ABCB4 wild-type clone and corresponding mutants. No differences of expression were observed between wild-type and mutant gene products. Instead, both mutations caused a reduction of phosphatidylcholine secretion compared with the wild-type transfected cell lines. On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile.

The aim of this experiment was to assay astronaut antioxidant status, to analyse red blood cell membrane composition of astronauts prior and after flight and to study the correlation with oxidative stress that erythrocytes have undergone due to space radiations. Results obtained from this single case study, indicate that during a short time flight, erythrocytes decrease their antioxidant defences, to counteract oxidative stress.

To test the effects of low levels of radiation from space on living organisms, we flew Xenopus laevis embryos at different stages of development on a stratospheric balloon (BI.R.BA mission). After recovery, different parameters were analyzed to assess the effects of flight, with particular regard to oxidative stress damage. Because of failed temperature control during flight, the flight shielded embryos (FC) could not be used for biochemical or morphological comparisons. In contrast, the incubation conditions (i.e. temperature, containers, volumes) for the flight embryos (F) were parallel to those for the ground controls. Mortality data show that younger embryos (16 h) flown on the balloon (F) are more sensitive to radiation exposure than older ones (40 h and 6 days). Exposure during flight lowered the antioxidant potential in all embryos, particularly older ones. These preliminary data demonstrate that flight on a stratospheric balloon might affect antioxidant metabolism, though it is not yet possible to correlate these results with low radiation exposure during flight.

Reactive oxygen species (ROS) are formed and degraded in all aerobic organisms, but their role during embryonic development has not yet been well established. In this paper, we report the activities of various enzymes involved in antioxidant metabolism during the first 7 days of embryonic development of Xenopus laevis embryos. During the first two days of development, embryo antioxidant metabolism is based on catalase and superoxide dismutase activities. Later, the glutathione system is activated, and the activity of all the enzymes involved increases. The results presented in this study, together with previously reported data, support the hypothesis that antioxidant defences may include enzymes that are genetically regulated, while the other systems that appear to be environmentally modulated become relevant later in development, probably to protect embryos from environmental and toxic factors.

Background Diets and Omega-3 polyunsaturated fatty acids have been considered as important factors to reduce the risk of cardiovascular and inflammatory diseases, but there are few details on the effects on healthy subjects. The aim of the present study was to examine the variation of several physiological parameters in healthy subjects on different diets supplemented with Omega-3 fatty acids. Materials and methods The experiment was carried out on 33 subjects divided into four groups according to a double-blind cross-over design with a 1 : 1 ratio for Omega-3 (vs. placebo) and open-label parallel-groups with a 1 : 1 ratio for the Zone diet (vs. the diet suggested by the Italian National Research Institute for Nutrition and Foods). Blood samples were collected at the beginning of the experiment and after 35 (cross-over) and 70 days. The Profile of Mood States test (POMS) was also performed. Results The arachidonic acid/eicosapentaenoic acid ratio (AA/EPA) was strongly reduced by Omega-3 with a supplementary effect of the diet and in particular the Zone diet. The AA/EPA reduction was correlated with a concomitant decrease of insulin and homocysteine levels. The Zone diet reduced skinfold thickness and body fat percentage and also showed antioxidant effects. The mood state changed after Omega-3 supplementation, with an increased POMS index. This was related to a concomitant reduction of AA/EPA and was particularly evident in the Zone diet. Conclusion AA/EPA and mood state are differently influenced by diet and Omega-3, body fat is particularly reduced by Zone diet, while blood parameters such as triglycerides/HDL ratio, insulin and homocysteine are related to AA/EPA variations. These findings are discussed in terms of differences in the composition of the diets and the influences of Omega-3 on physiological functions.

The TARDIKISS (Tardigrades in Space) experiment was part of the Biokon in Space (BIOKIS) payload, a set of multidisciplinary experiments performed during the DAMA (Dark Matter) mission organized by Italian Space Agency and Italian Air Force in 2011. This mission supported the execution of experiments in short duration (16 days) taking the advantage of the microgravity environment on board of the Space Shuttle Endeavour (its last mission STS-134) docked to the International Space Station. TARDIKISS was composed of three sample sets: one flight sample and two ground control samples. These samples provided the biological material used to test as space stressors, including microgravity, affected animal survivability, life cycle, DNA integrity, and pathways of molecules working as antioxidants. In this paper we compared the molecular pathways of some antioxidant molecules, thiobarbituric acid reactive substances, and fatty acid composition between flight and control samples in two tardigrade species, namely,Paramacrobiotus richtersiandRamazzottius oberhaeuseri. In both species, the activities of ROS scavenging enzymes, the total content of glutathione, and the fatty acids composition between flight and control samples showed few significant differences. TARDIKISS experiment, together with a previous space experiment (TARSE), further confirms that both desiccated and hydrated tardigrades represent useful animal tool for space research.

1 alpha, 25-dihydroxyvitamin D3 was previously shown to induce cell death in brain tumour cell lines when added to the medium at micromolar concentration. In this paper we show that Cholecalciferol, a poor ligand of the vitamin D receptor, also induces cell death of HU197 human glioblastoma cell line and early passages cultures derived from a recurrent human glioblastoma. This finding suggests that the effects of vitamin D metabolites on brain tumour cells are at least partially independent from the activation of the classic nuclear receptor pathway. Vitamin D metabolites have been shown to activate the sphingomyelin pathway inducing an increase in cellular ceramide concentration. We determined the levels of sphingomyelin ceramide and ganglioside GD3 in Hu197 cells after treatment with cholecalciferol. A significant increase in ceramide concentration and a proportional decrease in sphingomyelin was already present after 6 hours of cholecalciferol treatment when no morphological changes were visible in the cultures. Treatment with ceramides (N-acetylsphingosine or natural ceramide from bovine brain) of the same cells also induces cell death. Similarly, treatment of the same cells with bacterial Sphingomyelinase also results in cell death. The demonstration of an increase in intracellular ceramide after cholecalciferol treatment and the ability of ceramide to induce cell death suggest that the sphingomyelin pathway may be implicated in the effect of vitamin D metabolites on human glioblastoma cells. Inhibition of ceramide biosynthesis by fumonisin B1 treatment did not alter the dose response curve of HU197 cells to cholecalciferol. Insensitivity to fumonisin B1 together with a decrease in sphingomyelin content after cholecalciferol treatment indicate that activation of sphingomyelinase should be responsible for the increase in intracellular ceramide concentration.

Adverse health effects of air pollution attributed mainly to airborne particulate matter have been well documented in the last couple of decades. Short term exposure, referring to a few hours exposure, to high ambient PM10 concentration is linked to increased hospitalization rates for cardiovascular events, typically 24 h after air pollution peaks. Particulate matter exposure is related to pulmonary and cardiovascular diseases, with increased oxidative stress and inflammatory status. Previously, we have demonstrated that repeated intratracheal instillation of PM10sum in BALB/c mice leads to respiratory tract inflammation, creating in lung a condition which could potentially evolve in a systemic toxic reaction. Additionally, plasma membrane and tissue lipids are easily affected by oxidative stress and directly correlated with inflammatory products. With this aim, in the present investigation using the same model, we analyzed the toxic potential of PM10sum exposure on lipid plasma membrane composition, lipid peroxidation and the mechanisms of cells protection in multiple organs such as lung, heart, liver and brain. Obtained results indicated that PM10 exposure led to lung lipid reshaping, in particular phospholipid and cholesterol content increases; concomitantly, the generation of oxidative stress caused lipid peroxidation. In liver we found significant changes in lipid content, mainly due to an increase of phosphatidylcholine, and in total fatty acid composition with a more pronounced level of docosahexaenoic acid; these changes were statistically correlated to lung molecular markers. Heart and brain were similarly affected; heart was significantly enriched in triglycerides in half of the PM10sum treated mice. These results demonstrated a direct involvement of PM10sum in affecting lipid metabolism and oxidative stress in peripheral tissues that might be related to the serious systemic air-pollution effects on human health.

The effect of a 4-week cthanol administration on: (1) glycoprotcin content of brush border membrane (BBM): (2) galactosyltransferasc activity: (3) hpid composition and fluidity ol intestinal microsomes prepared from young and adult rats was investigated. In spite of a lower alcohol consump- tion, the more dramatic effects of treatment have been observed in the older rats, where BBM protein- bound hexoses and microsomal galactosyltransferasc activity were significantly decreased. On the contrary, these parameters were unaffected in young rats. However, both rat groups were similarly affected in having their microsomal cholesterol contents significantly increased. Microsomal membranes from ethanol-fed adult rats were less fluid compared to control rats: the high fluorescence anisotropy value could be related to the high cholesterol/phospholipid ratio and to the decrease of the unsaturated fattv acids C22:4 and C22:6.

In a sample of meningosarcoma, obtained at the time of surgery, the amount of total gangliosides and phospholipids was examined, together with the cholesterol content and the distribution of different ganglioside and phospholipid species. The phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4, 5-bisphosphate and phosphatidylcholine fatty acid composition was also analyzed. The ganglioside pattern in the meningosarcoma was different from the previously reported pattern in meningiomas of different histological origin, showing a higher concentration of GD3, indicating that the so-called b pathway of ganglioside biosynthesis was the preferred one in this type of tumor; moreover the percentage content of polysialylated gangliosides was very low. Cholesterol and phospholipid content was lower than in meningiomas; the phosphatidylcholine increase and the sphingomyelin decrease would indicate a lower membrane microviscosity, a characteristic of tumor cells. Phosphoinositide and phosphatidylcholine fatty acid analysis revealed a considerable amount of docosahexaenoic acid. This abnormal presence of this fatty acid could lead to the production, after receptor stimulation, of a diacylglycerol containing docosahexaenoic acid, which, in turn, could be responsible for an altered activation pattern of protein kinase C, in this way promoting carcinogenesis.

Background PUFAs are important molecules for membrane order and function; they can modify inflammation-inducible cytokines production, eicosanoid production, plasma triacylglycerol synthesis and gene expression. Recent studies suggest that n-3 PUFAs can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer therapy. N-3 PUFAs could alter cancer growth influencing cell replication, cell cycle, and cell death. The question that remains to be answered is how n-3 PUFAs can affect so many physiological processes. We hypothesize that n-3 PUFAs alter membrane stability, modifying cellular signalling in breast cancer cells. Methods Two lines of human breast cancer cells characterized by different expression of ER and EGFR receptors were treated with AA, EPA or DHA. We have used the MTT viability test and expression of apoptotic markers to evaluate the effect of PUFAs on cancer growth. Phospholipids were analysed by HPLC/GC, to assess n-3 incorporation into the cell membrane. Results We have observed that EPA and DHA induce cell apoptosis, a reduction of cell viability and the expression of Bcl2 and procaspase-8. Moreover, DHA slightly reduces the concentration of EGFR but EPA has no effect. Both EPA and DHA reduce the activation of EGFR. N-3 fatty acids are partially metabolized in both cell lines; AA is integrated without being further metabolized. We have analysed the fatty acid pattern in membrane phospholipids where they are incorporated with different degrees of specificity. N-3 PUFAs influence the n-6 content and vice versa. Conclusions Our results indicate that n-3 PUFA feeding might induce modifications of breast cancer membrane structure that increases the degree of fatty acid unsaturation. This paper underlines the importance of nutritional factors on health maintenance and on disease prevention.

All living organisms are constantly exposed to oxidant agents deriving from both endogenous and exogenous sources capable to modify biomolecules and induce damages. Free radicals generated by oxidative stress exert an important role in the development of tissue damage and aging. Reactive species (RS) derived from oxygen (ROS) and nitrogen (RNS) pertain to free radicals family and are constituted by various forms of activated oxygen or nitrogen. RS are continuosly produced during normal physiological events but can be removed by antioxidant defence mechanism: the imbalance between RS and antioxidant defence mechanism leads to modifications in cellular membrane or intracellular molecules. In this chapter only endogenous antioxidant molecules will be critically discussed, such as Glutathione, Alpha-lipoic acid, Coenzyme Q, Ferritin, Uric acid, Bilirubin, Metallothioneine, L-carnitine and Melatonin.

The most frequent drawback of doxorubicin is the onset of drug resistance, due to the active efflux through P-glycoprotein (Pgp). Recently formulations of liposome-encapsulated doxorubicin have been approved for the treatment of tumors resistant to conventional anticancer drugs, but the molecular basis of their efficacy is not known. To clarify by which mechanisms the liposome-encapsulated doxorubicin is effective in drug-resistant cancer cells, we analyzed the effects of doxorubicin and doxorubicin-containing anionic liposomal nanoparticles ("Lipodox") on the drug-sensitive human colon cancer HT29 cells and on the drug-resistant HT29-dx cells. Interestingly, we did not detect any difference in drug accumulation and toxicity between free doxorubicin and Lipodox in HT29 cells, but Lipodox was significantly more effective than doxorubicin in HT29-dx cells, which are rich in Pgp. This effect was lost in HT29-dx cells silenced for Pgp and acquired by HT29 cells overexpressing Pgp. Lipodox was less extruded by Pgp than doxorubicin and inhibited the pump activity. This inhibition was due to a double effect: the liposome shell per se altered the composition of rafts in resistant cells and decreased the lipid raft-associated amount of Pgp, and the doxorubicin-loaded liposomes directly impaired transport and ATPase activity of Pgp. The efficacy of Lipodox was not increased by verapamil and cyclosporin A and was underwent interference by colchicine. Binding assays revealed that Lipodox competed with verapamil for binding Pgp and hampered the interaction of colchicine with this transporter. Site-directed mutagenesis experiments demonstrated that glycine 185 is a critical residue for the direct inhibitory effect of Lipodox on Pgp. Our work describes novel properties of liposomal doxorubicin, investigating the molecular bases that make this formulation an inhibitor of Pgp activity and a vehicle particularly indicated against drug-resistant tumors.

Recent observations showed that long chain omega 3 polyunsaturated fatty acids (n-3 LCPUFA) could represent a potential treatment for elderly depression. To determine if a n-3 LCPUFA containing supplement improves depressive symptoms, changes phospholipids acids profile and ameliorates Health related quality of life (HRQoL) in depressed elderly patients.Two-months, randomized, double-blind, placebo-controlled trial.Nursing home in Pavia, Italy.Forty-six depressed females, aged 66-95 years.22 depressed females were included in the intervention group (n-3 group, that received 2.5 g/day of n-3 LCPUFA, with 1.67 grams of EPA and 0.83 grams of DHA), and 24 patients in the placebo group. The primary endpoint was the improvement of depressive symptoms as evaluated by Geriatric Depression Scale (GDS). Secondary endpoints were the evaluation of modifications of erythrocyte membrane phospholipids fatty acid profile and of of HRQoL, by using the Short-Form 36-Item Health Survey (SF-36). All parameters were assessed before and after the treatment period of 8 weeks.The mean GDS at 2 months was significantly lowered only for the n-3 group. SF-36 physical and mental components were significantly increased in the intervention group. Compliance was good, as confirmed by erythrocyte membrane phospholipid FA concentrations, with significant increase of EPA and DHA in the intervention group.The supplementation of n-3 LCPUFA in elderly female patients reduces the occurrence of depressive symptoms, improves phospholipids fatty acids profile and health-related quality of life.

Oxidative stress is strictly correlated to the pathogenesis of many diseases, and a diet rich in fruits and vegetables, or adequately integrated, is currently considered to be a protective and preventive factor. This study aimed to analyze the efficacy of a 1 h preincubation with the highest nontoxic dose of a characterized Mentha longifolia extract (80 μg/mL) in protecting human keratinocytes (NCTC2544) from chemically induced oxidative stress (500 μM H2O2 for 2, 16, and 24 h). As reference synthetic pure compounds rosmarinic acid (360.31 μg/mL), a major mint phenolic constituent, and resveratrol (31.95 mg/mL), a well-known antioxidant, were used. Cellular viability was significantly protected by mint, which limited protein and DNA damage, decreased lipid peroxidation, and preserved glutathione and superoxide dismutase activity in the shorter phases of oxidative stress induction, in extents comparable to or better than those of pure compounds. These data suggest that mint use as only a flavoring has to be revised, taking into consideration its enrichment in foodstuff and cosmetics.

All living organisms are constantly exposed to oxidant agents deriving from both endogenous and exogenous sources capable to modify biomolecules and induce damages. Free radicals generated by oxidative stress exert an important role in the development of tissue damage and aging. Reactive species (RS) derived from oxygen (ROS) and nitrogen (RNS) pertain to free radicals family and are constituted by various forms of activated oxygen or nitrogen. RS are continuosly produced during normal physiological events but can be removed by antioxidant defence mechanism: the imbalance between RS and antioxidant defence mechanism leads to modifications in cellular membrane or intracellular molecules. In this chapter only endogenous antioxidant molecules will be critically discussed, such as Glutathione, Alpha-lipoic acid, Coenzyme Q, Ferritin, Uric acid, Bilirubin, Metallothioneine, L-carnitine and Melatonin.

Five young patients with Niemann-Pick disease type B were treated with repeated implantations of amniotic epithelial cells, as a source of exogenous sphingomyelinase. This treatment abolished the recurrent infections, mainly of the respiratory tract, and led to other improvements of the general conditions of the patients. In particular, we noticed a disappearance of vomiting, a recovery from muscular hypotrophy, and significantly reduced pulmonary distress. In four subjects, who were in a prepuberal state, there was a puberal spurt with a concomitant burst of growth. In two cases, characterized by a greater than normal content of sphingomyelin in urinary sediments, a single implantation caused a sustained normalization of sphingomyelin and total phospholipids in the urine. Finally, sphingomyelinase activity of peripheral leukocytes, when assayed 0.5 to 4 months after some of the implantations, showed a rise to heterozygous values in 30-40% of the assays.

Reactive oxygen species (ROS) are formed in all aerobic organisms, potentially leading to oxidative damage of all biological molecules. A number of defence mechanisms have developed to protect the organism from attack by ROS. Desiccation tolerance is correlated with an increase in the antioxidant potential in several organisms, but the regulation of the antioxidant defence system is complex and its role in desiccation-tolerant organisms is not yet firmly established. To determine if anhydrobiotic tardigrades have an antioxidant defence system, capable of counteracting ROS, we compared the activity of several antioxidant enzymes, the fatty acid composition and Heat shock protein expression in two physiological states (desiccated vs. hydrated) of the tardigrade Paramacrobiotus richtersi. In hydrated tardigrades, superoxide dismutase and catalase show comparable activities, while in desiccated specimens the activity of superoxide dismutase increases. Both glutathione peroxidase and glutathione were induced by desiccation. The percentage of fatty acid composition of polyunsaturated fatty acids and the amount of thiobarbituric acid reactive substances are higher in desiccated animals than in hydrated ones. Lastly, desiccated tardigrades did not differ significantly from the hydrated ones in the relative levels of Hsp70 and Hsp90. These results indicate that the possession of antioxidant metabolism could represent a crucial strategy to avoid damages during desiccation in anhydrobiotic tardigrades.

Space flights cause a number of patho-physiological changes. Oxidative damage has been demonstrated in astronauts after space flights. Oxidative stress is due to an imbalance between production of oxidant and antioxidative defence. In embryos of Xenopus laevis, the glutathione system is an inducible antioxidant defence. For this reason, we investigated the effect of gravity deprivation on endogenous antioxidant enzymes in X. laevis embryos developed for 6 days in a Random Positioning Machine. The results show that glutathione content and the activity of antioxidant enzymes increase in RPM embryos, suggesting the presence of a protective mechanism. An induction of antioxidant defence might play an important role for animals to adapt to micro-gravitational stress, possibly during actual space flights.

Although several studies have aimed to identify mare's milk proteins, only the major whey proteins and some caseins have yet been characterized. Incomplete sequencing of the equine genome and the difficulty of recovering highly hydrophobic proteins mean that little is known to date about the proteins associated with milk fat globules, which have been shown to play an important role in newborns' defense mechanisms. The fat fraction, in particular the distribution of unsaturated fatty acids, has been more extensively studied, but complex lipids are only partially elucidated. This study reports a 2-DE approach combined with a powerful method for de novo protein sequencing, and quali-quantitative data on complex lipid composition determined by high performance TLC (HPTLC) and GC. The presence in mare's milk of long-chain highly unsaturated fatty acids, and the evidence of close similarity between equine and human milk fat globule membrane proteins, support the use of mare's milk for human nutrition.

Omega-3 and omega-6 long-chain polyunsaturated fatty acids (LCPUFAs), are crucial to brain development and function. Increasing evidence indicates that deficiencies or metabolic imbalances of these fatty acids might be associated with childhood developmental and psychiatric disorders including attention-deficit/hyperactivity disorder (ADHD). Omega-3 are often lacking on modern diets. Moreover preliminary evidences suggest that supplementation with omega-3 LCPUFAs, might help in the management of the ADHD linked behavioural and learning difficulties. However, few studies published to date have involved different populations, study designs, treatments and outcome results. Thus, further researches are required to assess the durability of the treatment effects, to determine optimal composition and dosages of the supplement and to develop reliable ways to identify patients that might have some benefits from this kind of treatment, also because the study of LCPUFAs and their metabolism might offer new approaches to the early identification and management of ADHD. In this paper, we provide new insight on the lipid pattern in plasma and red blood cells (RBC) phospholipids, together with evaluation of the arachidonic acid (AA)/eicosapentaenoic acid (EPA) ratio which seems to correlate with the improvement of the patients both from a biochemical and clinical point of view.

Heme (ferric protoporphyrin IX, FP) dissolves very rapidly into the lipid phase of membranes, and a large number of studies have focused attention on its possible toxic effect in whole cells or isolated membranes. However, because of its molecular structure and reactivity, different problems can be encountered during the course of studying biological samples containing FP. In this article, we discuss important interferences by FP and artifacts that can affect the experimental values. First, FP interferes with the Lowry's protein determination; therefore, membranes containing FP are overestimated in their protein content determined by this procedure. Second, freezing membranes at -20 degrees C artifactually increases the local concentration of FP, thereby enhancing FP-induced lipid peroxidation. Third, in the presence of thiol compounds such as N-acetyl cysteine, FP is degraded to products that interfere with the thiobarbituric acid assay, one of the most widely used methods to measure the extent of lipoperoxidation.

The lipid composition or the liver, spleen, brain, cerebellum and cerebrospinal fluid of a Gaucher disease type II patient who died at the age of 5 months was examined. The glycolipid analysis demonstrated a marked increase of total amounts not only in the peripheral tissues but also in the brain cerebellum and cerebrospinal fluid, with a prevalence of glucosylceramide. A reduction in gangliosides was observed in all the analysed tissues with a relative increase of GD3 in the nervous tissue. The fatty acid composition of glucosylceramide showed a prevalence of stearic acid in the central nervous system, while in the peripheral tissues palmitic acid was prevalent. This result suggests a different origin of the glucosylceramide stored in different tissues. The generalized reduction of gangliosides and their modified distribution together with the central nervous system GD3 increment represent a new observation. These data could be useful in the effort to clarify the pathophysiological mechanism of brain damage in neuronopathic Gaucher disease.

— Exposure to ethanol is known to alter cell membranes both chemically and biophysically; these effects may be related to the development of tolerance and physical dependence. In the present study, the effects of various concentrations of ethanol (1–250 mM) on the lipid composition after the first 6 days of Xenopus embryo development were investigated, using an in vitro fertilization technique. Lipid analysis revealed: (1) a decrease of the cholesterol/phospholipid molar ratio mainly derived from a higher content of phospholipids; (2) an increase of phospholipid unsaturated fatty acids, especially C20:4 and C20:5, with ethanol concentration of 150–250 mM; (3) a decrease of lipid-bound sialic acid with ethanol concentrations of ≥5 mM. These results underline that sialoglycoconjugates are a more sensitive target of alcohol in comparison with other lipid components. The cultured embryo method certainly represents a useful model for investigation of the direct effects of ethanol on lipid metabolism, excluding maternal interference which can lead to misinterpretation of data.

Erythrocytes, from 2- and 7-month-old rats chronically fed with a liquid diet containing ethanol, were analyzed for their susceptibility to lipid peroxidation estimated as thiobarbituric acid reactive substance (TBARS) products. In spite of a lower alcohol consumption, adult rats were the more affected by the ethanol treatment. Erythrocyte membranes prepared from alcoholic animals were more sensitive to lipid peroxidation than those prepared from control rats. In both age groups lipid analysis revealed similar changes: 1) an increased cholesterol/phospholipid molar ratio mainly derived from a higher content of cholesterol that accounts for the lower membrane fluidity and the higher tolerance to the disordering effect exerted by ethanol in vitro; 2) an increase of phospholipid unsaturated fatty acids, especially C20:4; 3) a modification of the phospholipid pattern, characterized in the ethanol group by an increase of PE and decrease of PE and decrease of PC levels; moreover, significant increases of the anionic phospholipids were detected in the adult group.

The aim of our study was to determine whether the minor polar components of virgin olive oil could have favorable effects (1) on fasting and postprandial lipid profile and (2) on low-density lipoprotein (LDL) composition and susceptibility to oxidation in vitro. Ten normolipidic subjects were included in a crossover study (two diet periods of 3 weeks) and received either virgin olive oil (OO diet) or oleic acid rich sunflower oil. An oral fat load was performed at the end of each period. The plasma lipid levels were not significantly different after both diets in the fasting and postprandial states. A few minor variations of the LDL composition were observed only in the postprandial lipemia, and they were different after both diets. The LDL oxidation susceptibility was evaluated by the formation of conjugated dienes. With LDL isolated in the fasting state, the diene production decreased (p = 0.0573) only after the OO diet. The dienes determined at time 0 and the maximal dienes obtained during the oxidation reaction decreased (p = 0.0145 and p = 0.0184, respectively) only after the OO fat load. Nevertheless, the diene production decrease was not significant (p = 0.0848). Our results suggest a mild effect of minor components of virgin olive oil related to a decrease of LDL susceptibility to oxidation; further analyses are necessary to give clear conclusions about their role.

The phospholipid profile of different chick embryo brain regions was studied from 11 to 21 days of development, revealing interesting changes in content and distribution. Total phospholipid phosphorus (P), in micrograms of P per microgram of DNA, increases significantly during development of cerebral hemispheres (CHs), optic lobes (OLs), and brainstem (BS). Compared with CH and OL, the BS shows at all stages a significantly higher concentration of phospholipid P, which in contrast decreases in the cerebellum (CB) during development. Moreover, the data show interesting differences between the right and the left portion of the brain. The distribution of phospholipid P and the fatty acid composition of phospholipids were asymmetric between left and right OL and CH, as were the concentrations of DNA and cholesterol, demonstrating lateralized neurochemical development in these structures, i.e., left OL, right OL, left CH, and right CH. The data are discussed also in relation to the potential importance of neurochemical lateralization for determining lateralized embryonic and postnatal behavior of this species.

Background Depression is one of the most frequently missed diagnoses in elderly people, with obvious negative effects on quality of life. Various studies have shown that long chain omega-3 polyunsaturated fatty acids (n-3 PUFA) may be useful in its management. Our objective was to evaluate whether a supplement containing n-3 PUFA improves depressive symptoms in depressed elderly patients, and whether the blood fatty acid pattern is correlated with these changes. Methods The severity of depressive symptoms according to the Geriatric Depression Scale (GDS), blood fatty acid composition and erythrocyte phospholipids were analyzed in 46 depressed females aged 66-95y, diagnosed with depression according to DSMIV, within the context of a randomized, double-blind, placebo-controlled trial. 22 depressed females were included in the intervention group (2.5 g/day of n-3 PUFA for 8 weeks), and 24 in the placebo group. We also measured immunological parameters (CD2, CD3, CD4, CD8, CD16, CD19 and cytokines (IL-5, IL-15). Results The mean GDS score and AA/EPA ratio, in whole blood and RBC membrane phospholipids, were significantly lower after 2 months supplementation with n-3 PUFA. A significant correlation between the amelioration of GDS and the AA/EPA ratio with some immunological parameters, such as CD2, CD19, CD4, CD16 and the ratio CD4/CD8, was also found. Nevertheless, omega-3 supplementation did not significantly improve the studied immunological functions. Conclusions n-3 PUFA supplementation ameliorates symptoms in elderly depression. The n-3 PUFA status may be monitored by means of the determination of whole blood AA/EPA ratio.

In this paper a method is presented which is suitable for the extraction, purification and analysis of serum gangliosides. The advantage in comparison with other previously published procedures is the complete extraction of sialoglycolipids without contamination of sialoglycoproteins and/or sialoglycopeptides. The method could be used as a second-level test for the diagnosis and follow-up of cancer patients, and also could be potentially used for pharmaco-kinetic studies after ganglioside treatment.

Background Nowadays no researches has been performed on fatty acid profile (FA) and desaturase activity in metabolically healthy obesity (MHO). The aim of this study was to assessed gender and BMI-related difference in FA, estimated desaturase activities and the efficacy on metabolic changes produced by 2-months well-balance diet in MHO subjects. Methods In 103 MHO subjects (30/73 M/F; age:42.2 ± 9.5) FA, estimated desaturase activity, body composition (by DXA), Body Mass Index (BMI), lipid profile, adipokines (leptin, adiponectin, grelin, glucagon-like peptide-1), insulin resistence (by Homestasis metabolic assessment), C-reactive proteine, Atherogenic index of plasma (AIP) and Body Shape Index (ABSI) have been assessed. Gender and BMI related difference have been evaluated and the efficacy produced by 2-months well-balance diet has been considered. Results At baseline, obese subjects, compared to overweight, show a significantly higher oleic (p

Background The activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors. Methods We studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells. Results MDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells. Conclusions Our work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells.

Background Omega-3 and -6 polyunsaturated fatty acids (LCPUFA), are important for good health conditions. They are present in membrane phospholipids. The ratio of total n-6:n-3 LCPUFA and arachidonic acid:eicosapentaenoic acid (AA and EPA), should not exceed 5:1. Increased intake of n-6 and decreased consumption of n-3 has resulted in much higher, ca 10/15:1 ratio in RBC fatty acids with the possible appearance of a pathological "scenario". The determination of RBC phospholipid LCPUFA contents and ratios is the method of choice for assessing fatty acid status but it is labour intensive and time consuming. Aims of the study [i] To describe and validate a rapid method, suitable for large scale population studies, for total blood fatty acid assay; [ii] to verify a possible correlation between total n-6:n-3 ratio and AA:EPA ratios in RBC phospholipids and in whole-blood total lipids, [iii] to assess usefulness of these ratio as biomarkers of LCPUFA status. Methods [1] Healthy volunteers and patients with various pathologies were recruited. [2] Fatty acid analyses by GC of methyl esters from directly derivatized whole blood total lipids and from RBC phospholipids were performed on fasting blood samples from 1432 subjects categorised according to their age, sex and any existing pathologies. AA:EPA ratio and the total n-6:n-3 ratio were determined. Results AA:EPA ratio is a more sensitive and reliable index for determining changes in total blood fatty acid and it is correlated with the ratio derived from extracted RBC phospholipids. Conclusions The described AA:EPA ratio is a simple, rapid and reliable method for determining n-3 fatty acid status.