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International audience; The present study provides the fatty acid, tocopherol, phytosterol, and polyphenol profiles of some Mediterranean oils extracted from pumpkin, melon, and black cumin seed oils and those of dietary argan seed oil. Gas chromatography analysis revealed that oleic and linoleic acids were the most abundant fatty acids. Argan and melon seed oils exhibited the highest levels of oleic acid (47.32±0.02%) and linoleic acid (58.35±0.26%), respectively. In terms of tocopherols, melon seed oil showed the highest amount (652.1±3.26 mg/kg) with a predominance of γ-tocopherol (633.1±18.81 mg/kg). The phytosterol content varied between 2237.00±37.55 µg/g for argan oil to 6995.55±224.01 µg/g for melon seed oil. High Performance Liquid Chromatography analysis also revealed the presence of several polyphenols: vanillin (0.59 mg equivalents Quercetin/100 g) for melon seed oil, and p-hydroxycinnamic acid (0.04 mg equivalents Quercetin/100 g), coumarine (0.05 mg equivalents Quercetin/100 g), and thymoquinone (1.2 mg equivalents Quercetin/100 g) for black cumin seed oil. The "Kit Radicaux Libres" (KRL) assay used to evaluate the scavenging properties of the oils showed that black cumin seed oil was the most efficient. On the light of the richness of all Mediterranean oil samples in bioactive compounds, the seed oils studied can be considered as important sources of nutrients endowed with cytoprotective properties which benefits in preventing age-related diseases which are characterized by an enhanced oxidative stress.

Background Incomplete vascularization of the retina in preterm infants carries a risk of retinopathy of prematurity (ROP). Progress in neonatal resuscitation in developing countries has led to the survival of an increasing number of premature infants, resulting in an increased rate of ROP and consequently in visual disability. Strategies to reduce ROP involve optimizing oxygen saturation, nutrition, and normalizing factors such as insulin-like growth factor 1 and n-3 long-chain polyunsaturated fatty acids (LC-PUFA). Our previous study, OmegaROP, showed that there is an accumulation or retention of docosahexaenoic acid (DHA) in mothers of infants developing ROP, suggesting abnormalities in the LC-PUFA placental transfer via fatty acid transporting proteins. The present study aims to better understand the LC-PUFA transport dysfunction in the fetoplacental unit during pregnancy and to find a novel target for the prevention of ROP development. Methods The study protocol is designed to evaluate the correlation between the expression level of placental fatty acid receptors and ROP occurrence. This ongoing study will include 100 patients: patients giving birth before 29 weeks of gestational age (GA) and patients with full-term pregnancies. Recruitment is planned for over 46 months. Maternal and cord blood samples as well as placental tissue samples will be taken following delivery. ROP screening will be performed using wide-field camera imaging according to the International Classification of ROP consensus statement. Discussion The results of this study will have a tangible impact on public health. Indeed, if we show a correlation between the expression level of placental omega-3 receptors and the occurrence of ROP, it would be an essential step in discovering novel pathophysiological mechanisms involved in this retinopathy. Trial registration: 2020-A03253-36

The aim of this study was to evaluate docosahexaenoic acid (DHA) as a potential antifibrotic agent after glaucoma filtration surgery (GFS) in rats.A total of 36 10-week-old Brown Norway rats underwent GFS. Animals were equally divided into three groups: a control group, a DHA group and a mitomycin C (MMC) group. Intraocular pressure (IOP) was measured using a dynamic rebound tonometer, and a photograph of the surgical site was taken on days 1, 3, 7, 10, 14 and 17. The incorporation of DHA into fibroblasts was evaluated by gas chromatography. The expression of alfa-smooth muscle actin (α-SMA) and Smad proteins was assessed by Western blotting.IOP decreased after surgery in animals from the three groups on day 1 after surgery. Over time, IOP remained lower in the DHA and MMC groups than in the control group (median [interquartile range] 8.0 [7.0-8.0] and 8.0 [7.3-8.0] mmHg vs. 9.0 [8.0-9.0] mmHg, respectively; p 0.001). Bleb area in the DHA and MMC groups remained larger than that of the control group from day 7 to day 14 (3.9 [2.9-5.2] and 3.5 [2.3-4.4] mmThe impact of DHA on IOP and bleb area was similar to that of MMC. The mechanisms of action of DHA in rat eye fibroblasts deserve further investigation.

International audience; Spatial changes of fatty acids in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids, EPA-containing triglycerides, DHA-containing phospholipids, or DHA-containing triglycerides. Qualitative changes in fatty acid composition of plasma, erythrocytes and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) species in retina were determined by liquid chromatography coupled to tandem mass spectrometry and MALDI (Matrix Assisted Laser Desorption Ionization) coupled to mass spectrometry imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that phospholipids as well as triglycerides are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain polyunsaturated fatty acids (VLC-PUFAs, more than 28 carbons) following phospholipid-EPA and triglyceride-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of fatty acids in the rat retina.

Structure and function of the retina mainly rely on its fatty acid (FA) composition. Evidence from epidemiological studies and from animal experiments indicates that FA composition of the retina is influenced by the diet. Mice under chronic high-fat diet (HFD) develop metabolic syndrome, a risk factor for diabetes that is associated with structural and functional alterations of the retina. Here, we studied the impact of chronic exposure of mice to HFD on retinal FA composition. C57BL/6 J male mice were fed either a chow diet or a HFD for 11 weeks. As expected, HFD induced weight gain, adiposity, hyperglycemia and dyslipidemia. The retinal FA composition was determined by gas chromatography coupled to flame ionization detection. No significant change in the relative abundance of total saturated FAs (SFAs), total monounsaturated FAs (MUFAs) or total polyunsaturated FAs (PUFAs) was observed. However, retinas of HFD-fed mice displayed decreased amounts of C24:0 (p = 0.0231), C16:1n-7 (p < 0.0001), C18:1n-7 (p < 0.0001), C20:3n-9 (p = 0.0425) and C20:3n-6 (p = 0.0008), and an increased amount of C20:2n-6 (p < 0.0001). In addition, the ratio of linoleic acid (C18:2n-6) to alpha-linolenic acid (C18:3n-3) was increased in the retinas of HFD-fed mice (15.0 +/- 0.8 versus 11.8 +/- 0.6 in HFD and CD, respectively, p = 0.0045). No modification in the contents of arachidonic acid (C20:4n-6, AA) and docosahexaenoic acid (C22:6n-3, DHA) were observed. Analysis of dimethylacetals (DMA), which are residues of plasmalogens (Pls), revealed that the amount of Pls containing octadecanal-aldehydes (DMA C18:0) was significantly increased in HFD-fed mice (p = 0.0447). This increase was, at least in part, balanced by a decrease in Pls containing 7-octadecanal-aldehydes (DMA C18:1n-7) (p = 0.0007). In conclusion, HFD had an impact on the relative proportion of essential dietary fatty acids linoleic acid and alpha-linolenic acid that are incorporated in the retina. However, this imbalance in PUFA precursors did not alter the content of the two major retinal long-chain PUFAs, AA and DHA. HFD consumption also led to alterations in the retinal SFAs, MUFAs and Pls profiles.

Communication between neurons and glia plays a major role in nervous tissue homeostasis. It is thought to participate in tuning cholesterol metabolism to cellular demand, which is a critical issue for neuronal health. Cholesterol is a membrane lipid crucial for nervous tissue functioning, and perturbed regulation of its metabolism has been linked to several neurodegenerative disorders. In the brain, 24(S)-hydroxycholesterol (24S-OHC) is an oxysterol synthesized by neurons to eliminate cholesterol, and 24S-OHC has been shown to regulate cholesterol metabolism in astrocytes, glial cells which provide cholesterol to neurons. In the retina, 24S-OHC is also an elimination product of cholesterol produced by neurons, especially the retinal ganglion cells. However, it is not known whether Muller cells, the major macroglial cells of the retina, play the role of cholesterol provider for retinal neurons and whether they respond to 24S-OHC signaling, similarly to brain glial cells. In the present study, primary cultures of rat Muller cells were treated with 0, 0.5 or 1.5 μM 24S-OHC for 48 hours. The levels of cholesterol, precursors and oxysterols were quantified using gas chromatography coupled to flame-ionization detection or mass spectrometry. In addition, the expression of key genes related to cholesterol metabolism was analyzed using RTq-PCR. Muller cells were shown to express many genes linked to cholesterol metabolism, including genes coding for proteins implicated in cholesterol biosynthesis (HMGCR), cholesterol uptake and export via lipoproteins (LDL-R, SR-BI, ApoE and ABACA1) and regulation of cholesterol metabolism (SREBP2 and LXRβ). Cholesterol and several of its precursors and oxidative products were present. CYP27A1, the main retinal enzyme implicated in cholesterol elimination via oxysterol production, was quantified at low transcript levels but neither of its two typical products were detected in Muller cells. Furthermore, our results demonstrate that 24S-OHC has a strong hypocholesterolemic effect in Muller cells, leading to cholesterol depletion (-37 % at 1.5 μM). This was mediated by a decrease in cholesterol synthesis, as illustrated by reduced levels of cholesterol precursors: desmosterol (-38 % at 1.5 μM) and lathosterol (-84 % at 1.5 μM), and strong downregulation of HMGCR gene expression (2.4 fold decrease at 1.5μM). In addition, LDL-R and SR-BI gene expression were reduced in response to 24S-OHC treatment (2 fold and 1.6 fold at 1.5 μM, respectively), suggesting diminished lipoprotein uptake by the cells. On the contrary, there was a dramatic overexpression of ABCA1 transporter (10 fold increase at 1.5 μM), probably mediating an increase in cholesterol efflux. Finally, 24S-OHC induced a small but significant upregulation of the CYP27A1 gene. These data indicate that Muller cells possess the necessary cholesterol metabolism machinery and that they are able to sharply adjust their cholesterol metabolism in response to 24S-OHC, a signal molecule of neuronal cholesterol status. This suggests that Muller cells could be major players of cholesterol homeostasis in the retina via neuron-glia crosstalk.

International audience; Reward-processing impairment is a common symptomatic dimension of several psychiatric disorders. However, whether the underlying pathological mechanisms are common is unknown. Herein, we asked if the decrease in the n-3 polyunsaturated fatty acid (PUFA) lipid species, consistently described in these pathologies, could underlie reward-processing deficits. We show that reduced n-3 PUFA biostatus in mice leads to selective motivational impairments. Electrophysiological recordings revealed increased collateral inhibition of dopamine D2 receptor-expressing medium spiny neurons (D2-MSNs) onto dopamine D1 receptor-expressing MSNs in the nucleus accumbens, a main brain region for the modulation of motivation. Strikingly, transgenically preventing n-3 PUFA deficiency selectively in D2-expressing neurons normalizes MSN collateral inhibition and enhances motivation. These results constitute the first demonstration of a causal link between a behavioral deficit and n-3 PUFA decrease in a discrete neuronal population and suggest that lower n-3 PUFA biostatus in psychopathologies could participate in the etiology of reward-related symptoms.

Purpose Muller glial cells (MGC) are involved in the retinal homeostasis and they are sustaining cholesterol homeostasis. A lack or an excess of cholesterol in neurons can cause neuro-degeneration. Cholesterol is eliminated from the retina mainly as 24S-hydroxycholesterol (24S-OHC). 24S-OHC is produced by 24S-hydroxylase (CYP46A1) in retinal ganglion cells (RGC) and is overexpressed during glaucoma. the objective of this study was to determine whether 24S-OHC triggers MGC membrane dynamics involving lipid rafts and contribute to gliosis at early and late time points. Methods MGCs were grown in vitro from retinas of young rats. They were treated with 24S-OHC (10 μM) for 2 min or 6h. Lipid rafts of MGC membranes were obtained after 1% lubrol lysis and 20h-ultracentrifugation at 180,000 g in a sucrose gradient. The expression of caveolin-1, Flotillin-1, GFAP, Vimentin, Connexin-30, Connexin-43, phosphorylated and non phosphorylated p38 and p42-44 MAPK was analyzed by Western-blotting. High-performance liquid chromatography coupled with mass spectrometry (LC-MS) was used to estimate the levels of ganglioside GM3 (monosialodihexosylganglioside), and protein pathways were analyzed by nanoLC-MS/MS in raft and non raft fractions from MGCs after treatment with 24S-HOC. Results Cholesterol, sphingomyelin and saturated fatty acids C15:0, C16:0, C17:0 and C18:0 were enriched in the rafts fractions in MGCs. Structural proteins, Caveolin-1 and flotillin-1, and functional proteins, Connexin-30 and -43, were localized in the MGCs rafts. Ganglioside GM3 showed a characteristic enrichment in the raft fractions. Protein implicated in adhesion or oxidative stress pathways in the raft and non-raft fractions were up and down-regulated by treatment with 24S-OHC. Conclusions Our data showed that 24S-OHC induced early changes in protein distribution in raft microdomains.

PMCID: PMC5666899; International audience; Argan oil is widely used in Morocco in traditional medicine. Its ability to treat cardiovascular diseases is well-established. However, nothing is known about its effects on neurodegenerative diseases, which are often associated with increased oxidative stress leading to lipid peroxidation and the formation of 7-ketocholesterol (7KC) resulting from cholesterol auto-oxidation. As 7KC induces oxidative stress, inflammation and cell death, it is important to identify compounds able to impair its harmful effects. These compounds may be either natural or synthetic molecules or mixtures of molecules such as oils. In this context: (i) the lipid profiles of dietary argan oils from Berkane and Agadir (Morocco) in fatty acids, phytosterols, tocopherols and polyphenols were determined by different chromatographic techniques; and (ii) their anti-oxidant and cytoprotective effects in 158N murine oligodendrocytes cultured with 7KC (25-50 mu M; 24 h) without and with argan oil (0.1% v/v) or -tocopherol (400 mu M, positive control) were evaluated with complementary techniques of cellular and molecular biology. Among the unsaturated fatty acids present in argan oils, oleate (C18:1 n-9) and linoleate (C18:1 n-6) were the most abundant; the highest quantities of saturated fatty acids were palmitate (C16:0) and stearate (C18:0). Several phytosterols were found, mainly schottenol and spinasterol (specific to argan oil), cycloartenol, beta-amyrin and citrostadienol alpha- and gamma-tocopherols were also present. Tyrosol and protocatechic acid were the only polyphenols detected. Argan and extra virgin olive oils have many compounds in common, principally oleate and linoleate, and tocopherols. Kit Radicaux Libres (KRL) and ferric reducing antioxidant power (FRAP) tests showed that argan and extra virgin olive oils have anti-oxidant properties. Argan oils were able to attenuate the cytotoxic effects of 7KC on 158N cells: loss of cell adhesion, cell growth inhibition, increased plasma membrane permeability, mitochondrial, peroxisomal and lysosomal dysfunction, and the induction of oxiapoptophagy (OXIdation + APOPTOsis + autoPHAGY). Altogether, our data obtained in 158N oligodendrocytes provide evidence that argan oil is able to counteract the toxic effects of 7KC on nerve cells, thus suggesting that some of its compounds could prevent or mitigate neurodegenerative diseases to the extent that they are able to cross the blood-brain barrier.

International audience

Lipid peroxidation products, such as 7-ketocholesterol (7KC), may be increased in the body fluids and tissues of patients with neurodegenerative diseases and trigger microglial dysfunction involved in neurodegeneration. It is therefore important to identify synthetic and natural molecules able to impair the toxic effects of 7KC. We determined the impact of 7KC on murine microglial BV-2 cells, especially its ability to trigger mitochondrial and peroxisomal dysfunction, and evaluated the protective effects of alpha- and gamma-tocopherol, Trolox, and oleic acid (OA). Multiple complementary chemical assays, flow cytometric and biochemical methods were used to evaluate the antioxidant and cytoprotective properties of these molecules. According to various complementary assays to estimate antioxidant activity, only alpha-, and gamma-tocopherol, and Trolox had antioxidant properties. However, only alpha-tocopherol, gamma-tocopherol and OA were able to impair 7KC-induced loss of mitochondrial transmembrane potential, which is associated with increased permeability to propidium iodide, an indicator of cell death. In addition, alpha-and gamma-tocopherol, and OA were able to prevent the decrease in Abcd3 protein levels, which allows the measurement of peroxisomal mass, and in mRNA levels of Abcd1 and Abcd2, which encode for two transporters involved in peroxisomal beta-oxidation. Thus, 7KC-induced side effects are associated with mitochondrial and peroxisomal dysfunction which can be inversed by natural compounds, thus supporting the hypothesis that the composition of the diet can act on the function of organelles involved in neurodegenerative diseases.

The sterol profile of three Algerian virgin olive oils produced from Guasto, Blanquette and Rougette cultivar’s, varieties grown in the East of the country, was established by gas chromatography using a flame ionization detector and coupled to mass spectrometry. Four sterol compounds were identified and characterized in the three oils which are, sitosterol always predominant (43-66%), delta 5-avenasterol (8-13%), stigmasterol (≤ 1,5%) and campesterol (1-2%); one methyl sterol (4-methylsterol) : citrostadienol (4-7%) and two triterpenic alcohols : 24-methylene cycloartanol (10-33%) and cycloartenol (3-13%). These results vary according to campany and variety. Our study shows that most of these compounds were affected by climatic conditions especially during the campanies 2004-2005 and 2005-2006. The amounts of the four identified sterols are in agreement with the European regulation (Reg. (EEC) n° 2568/91, appendix 1).

Gangliosides make a wide family of glycosphingolipids ubiquitously expressed in mammalian tissues and particularly abundant in the brain and nervous system. They exhibit a huge diversity due to structural variations in both their oligosaccharidic chain and ceramide moiety, which represent a real analytical challenge. Since their discovery in the 1940s, methods have persistently improved until the emergence of Liquid Chromatography/Mass Spectrometry (LC/MS) which offers a high level of specificity and sensitivity and is suitable with high-throughput profiling studies. We describe here a comprehensive approach relying on various techniques and aiming at fully characterizing gangliosides in biological samples. First, total ganglioside content was determined by a biochemical assay. Second, ganglioside class composition was assessed by high performance thin layer chromatography followed by colorimetric revelation. Then, ceramide types of ganglioside classes were identified and their relative quantification was performed thanks to the development of a powerful and reliable LC/MS method. Finally, ceramides were structurally characterized and minor and less common ganglioside classes were identified using high resolution MS. These methods were applied to the rat retina to provide an exhaustive description of its ganglioside composition, giving the base for a better understanding of the precise roles of gangliosides in this tissue.

The retina is responsible for coding the light stimulus into a nervous signal that is transferred to the brain via the optic nerve. The retina is formed by the association of the neurosensory retina and the retinal pigment epithelium that is supported by Bruch’s membrane. Both the physical and metabolic associations between these partners are crucial for the functioning of the retina, by means of nutrient intake and removal of the cell and metabolic debris from the retina. Dysequilibrium are involved in the aging processes and pathologies such as age-related macular degeneration, the leading cause of visual loss after the age of 50 years inWestern countries. The retina is composed of several populations of cells including glia that is involved in cholesterol biosynthesis. Cholesterol is the main sterol in the retina. It is present as free form in cells and as esters in Bruch’s membrane. Accumulation of cholesteryl esters has been associated with aging of the retina and impairment of the retinal function. Under dietary influence and in situ synthesized, the metabolism of cholesterol is regulated by cell interactions, including neurons and glia via cholesterol-24S-hydroxylase. Several pathophysiological associations with cholesterol and its metabolism can be suggested, especially in relation to glaucoma and age-related macular degeneration., Cholestérol et pathologies oculaires : focus sur le rôle de la cholestérol-24S-hydroxylase dans l’homéostasie du cholestérol. La rétine est le tissu neurosensoriel de l’oeil qui assure la transduction visuelle, c’est-à-dire le codage de l’information lumineuse en influx nerveux. Le terme de rétine regroupe l’association de la rétine neurale et de l’épithélium pigmentaire rétinien qui repose sur la membrane de Bruch. L’association physique de ces deux partenaires et leurs interactions métaboliques sont des éléments indispensables au bon fonctionnement de la rétine, à la fois en termes d’apport en nutriments et de maintien de l’homéostasie. Des dérèglements de ces équilibres sont associés au vieillissement et à des pathologies comme la dégénérescence maculaire liée à l’âge, première cause de malvoyance après 50 ans dans les populations occidentales. La rétine neurale est composée de plusieurs types cellulaires, incluant la glie dont le rôle est crucial pour la biosynthèse du cholestérol dans la rétine. Le cholestérol est le principal stérol de la rétine. Il y est présent dans la cellule essentiellement en tant que cholestérol libre et d’esters de cholestérol au niveau extracellulaire dans la membrane de Bruch. L’accumulation de ces esters de cholestérol à ce niveau est un marqueur du vieillissement de la rétine et de la diminution de sa fonction. Sous influence alimentaire et synthétisé localement, le cholestérol est également régulé via les interactions entre populations cellulaires de la rétine, comme entre neurones et glie, via la cholestérol-24S-hydroxylase. Plusieurs liens physiopathologiques sont envisagés avec le cholestérol et son métabolisme en particulier avec les glaucomes ou la dégénérescence maculaire liée à l’âge.

In a recent study, we observed some oxyphytosterols in the plasma of healthy human subjects. This experiment was effected in order to determine if these compounds could be formed in vivo from phytosterols. Rats were fed with a high level of phytosterols (1% of the diet) and they were compared to rats deprived of phytosterols, using triglycerides purified from phytosterols. Their plasma were analysed for the main oxyphytosterols. The results show that sitostanetriol and campestanetriol were not formed in vivo from phytosterols. Their levels decreased during the experiment. The diet origin is highly probable for the compounds identified in human plasma. In particular, it seems that the sitostanetriol is eliminated very slowly from the organism.

A study was conducted to develop a new internal standard for the quantitative determination of oxyphytosterols. Tests on 5α-androsten-3β,17β-diol; 5α-androstan-3β, 17β-diol; 5-pregnen-3β,20α-diol; and 5α-pregnan-3β,20β-diolshowed that these compounds were not fully adequate. However, the compound 3β,22-dihydroxy-20-homo-5-pregnene, synthesized in 4 steps, resulted in a promising internal standard, with a molecule similar to hydroxysterols; retention time as trimethylsilyl in gas chromatography comprised between 5α-cholestane and 7α-hydroxycholesterol; clear mass spectrum in electronic impact mass spectrometry, with several intense ions suitable for selected ion monitoring-mass spectrometry. Further studies are necessary to observe the behavior of these compounds during the entire analytical procedure.

The oxidised derivatives of phytosterols (oxyphytosterols) were identified in plasma samples from thirteen healthy human volunteers, using MS. All the samples contained noticeable quantities of (24R)-5b,6b-epoxy-24-ethylcholestan-3b-ol (b-epoxysitostanol) and (24R)-ethylcholestan-3b,5a,6b-triol (sitostanetriol) and also trace levels of (24R)-5a,6a-epoxy-24-ethylcholestan-3b-ol (a-epoxysitostanol), (24R)-methylcholestan-3b,5a,6b-triol (campestanetriol) and (24R)-ethylch olest-5-en-3b-ol-7-one(7-ketositosterol). The amounts of these oxyphytosterols in plasma varied from 4·8 to 57·2 ng/ml. There are two possibilities concerning the origin of these compounds. First, they could come from the small amounts of oxyphytosterols in food. Second, they could originate from the in vivo oxidation of phytosterols in plasma. Very few data actually exist concerning these compounds. Their identification in human samples suggests that further research is necessary in this field. Oxyphytosterols: Phytosterols: Human plasma: Mass spectrometry

International audience; Gangliosides make a wide family of glycosphingolipids, highly heterogeneous in both the ceramide moiety and the oligosaccharide chain. While ubiquitously expressed in mammalian tissues, they are particularly abundant in the brain and the peripheral nervous system. Gangliosides are known to play a crucial role in the development, maintenance and functional integrity of the nervous system. However, the expression and roles of gangliosides in the retina, although often considered as a window on the brain, has been far less studied. We performed an in-depth analysis of gangliosides of the human retina, especially using powerful LC/MS methods. We compared the pattern of ganglioside classes and ceramide molecular species of this tissue with other ocular structures and with brain and plasma in elderly human individuals. About a hundred of ganglioside molecular species among 15 distinct classes were detected illustrating the huge structural diversity of these compounds. The retina exhibited a very diverse ganglioside profile and shared several common features with the brain (prominence of tetraosylgangliosides, abundance of d20:1 long chain base and 18:0 fatty acid...). However, the retina stood out with the specific expression of GD3, GT3 and AcGT3, which further presented a peculiar molecular species distribution. The unique ganglioside pattern we observed in the human retina suggests that these ganglioside species play a specific role in the structure and function of this tissue. This lipidomic study, by highlighting retina specific ganglioside species, opens up novel research directions for a better understanding of the biological role of gangliosides in the retina.

WOS:000320433100013 ; http://link.springer.com/journal/11746; International audience; Seeds from 12 Acacia cyanophylla ecotypes, harvested in Tunisia, were examined for their seed oil contents of carotenoids, tocopherols and phytosterols. The average carotenoid content (lutein and zeaxanthin) was ca. 102 mg kg(-1) of total extracted lipids. Lutein (ca. 97 mg kg(-1) of total extracted lipids) was usually more abundant than zeaxanthin (ca. 5 mg kg(-1) of total extracted lipids). The mean total tocopherol content was ca. 704 mg kg(-1) of total extracted lipids. The main isomer was alpha-tocopherol, with more than 75 % of total tocopherols (ca. 528 mg kg(-1) of total extracted lipids), followed by gamma-tocopherol (ca. 168 mg kg(-1) of total extracted lipids) and delta-tocopherol (ca. 86 mg kg(-1) of total lipids). High levels of phytosterols (ca. 7.8 g kg(-1) of total extracted lipids) were detected, among which beta-sitosterol was the most abundant (47 %). All these results highlight the richness of carotenoids, tocopherols and sterols in A. cyanophylla seed oil, and imply that this species might constitute a potential resource for the development of functional foods.

The aim of the present study was to identify and quantitatetrans isomers of C18 fatty acids in some French infant formulas. Twenty powdered infant formulas were purchased in pharmacies and supermarkets in order to assess theirtrans mono- and poly-unsaturated fatty acids content. The fatty acid profiles were examined using methyl and isopropyl ester derivatives. The combination of gas-liquid chromatography, high-performance liquid chromatography, and silver nitrate thin-layer chromatography was needed to describe the detailed fatty acid compositions of the samples, includingtrans isomers of unsaturated C18 fatty acids. All the samples containedtrans isomers of C18∶1 acid (mean level 1.97±0.28% of total fatty acids), with vaccenic acid being generally the major isomer (15 out of 20 samples), thus indicating the origin from bovine milk. All the formulas also contained various isomers of linoleic and α-linolenic acids, but at lower levels.Trans PUFA isomers are the same as those present in deodorized oils. In conclusion, all the infant formulas analyzed in this study contained sometrans fatty acids, including isomers of essential fatty acids. This should be taken into account in the dietary intake of the newborn.

Purpose Blood lipids are frequently used as a surrogate of lipid composition of peripheral tissues. Even if it is well accepted such a relationship has never been clearly demonstrated for the eye. The aim of this study was to determine in human samples whether a lipidomic approach based on red blood cells could reveal associations between circulating and ocular lipid profiles. Methods Red blood cells, retinas and optic nerves were collected from 9 human donors. The lipidomic analyses on these tissues consisted in gas chromatography and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS). Results Gas chromatographic approach did not shpw any relevant association between circulating and ocular lipids except for arachidonic acid whose circulating amounts were positively associated with its retinal and optic nerve levels. However, significant associations emerged from LC-ESI-MS analyses. Indeed, phospholipid species in red blood cells were positively or negatively associated with representative pools of retinal DHA (docosahexaenoic acid), retinal VLC-PUFA (very-long chain polyunsaturated fatty acids) or optic nerve plasmalogens. Conclusion First, our results show that LC-ESI-MS methodology is more appropriate than gas chromatography for lipidomics on red blood cells, and further extrapolation to ocular lipids. Second, this study has identified several individual lipid species as good candidates to represent circulating biomarkers of ocular lipids.

Purpose: Our previous studies suggested that CYP46A1 and 24S-hydroxycholesterol. (24SOH) may be associated with glaucoma. Loss of CYP46A1-expressing retinal. ganglion cells is involved in the activation of glia, and therefore possibly in the. disbalance of cholesterol. In this context, the purpose of our present work was to. emphasize the glial and longitudinal CYP46A1 expression after an interventional. glaucoma-related stress triggered by elevated intraocular pressure (IOP). Methods: Sprague–Dawley rats were submitted to laser photocoagulation of the. trabecular meshwork, limbus and episcleral veins in one eye to induce elevated IOP. Rats were euthanized at days 3, 14, 30 and 60 (n = 10 per time-point), and 24SOH. was measured in plasma and retina by gas chromatography–mass spectrometry. CYP46A1 was quantified by Western blotting. Glial activation was assessed by glial. fibrillary acidic protein immunoreactivity in Western blots and retinal cryosections. Results: Sustained high IOP was observed in experimental eyes from day 1 to day. 21. Plasma MCP-1 and ICAM-1, quantified using multiplex assay kits, were. increased at day 3. Glial activation was observed bilaterally at all time-points, at. lower levels in contralateral eyes than in experimental eyes. In experimental retinas,. CYP46A1 expression showed a transient increase at day 3 and then returned to. baseline. Plasma and retinal 24SOH peaked at day 14 and 30, respectively. Conclusions: These data show that CYP46A1 expression was induced early in. response to retinal stress but remained constant at late time-points, reinforcing the. constitutive role of CYP46A1 in maintaining cholesterol balance in neuronal tissues.

International audience; BACKGROUND: The assessment of blood lipids is very frequent in clinical research as it is assumed to reflect the lipid composition of peripheral tissues. Even well accepted such relationships have never been clearly established. This is particularly true in ophthalmology where the use of blood lipids has become very common following recent data linking lipid intake to ocular health and disease. In the present study, we wanted to determine in humans whether a lipidomic approach based on red blood cells could reveal associations between circulating and tissue lipid profiles. To check if the analytical sensitivity may be of importance in such analyses, we have used a double approach for lipidomics. METHODOLOGY AND PRINCIPAL FINDINGS: Red blood cells, retinas and optic nerves were collected from 9 human donors. The lipidomic analyses on tissues consisted in gas chromatography and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS). Gas chromatography did not reveal any relevant association between circulating and ocular fatty acids except for arachidonic acid whose circulating amounts were positively associated with its levels in the retina and in the optic nerve. In contrast, several significant associations emerged from LC-ESI-MS analyses. Particularly, lipid entities in red blood cells were positively or negatively associated with representative pools of retinal docosahexaenoic acid (DHA), retinal very-long chain polyunsaturated fatty acids (VLC-PUFA) or optic nerve plasmalogens. CONCLUSIONS AND SIGNIFICANCE: LC-ESI-MS is more appropriate than gas chromatography for lipidomics on red blood cells, and further extrapolation to ocular lipids. The several individual lipid species we have identified are good candidates to represent circulating biomarkers of ocular lipids. However, further investigation is needed before considering them as indexes of disease risk and before using them in clinical studies on optic nerve neuropathies or retinal diseases displaying photoreceptors degeneration.

Oxysterols, found in some commonly consumed foods, can induce a wide range of cytotoxic effects, which have been extensively studied. On the other hand, the side effects of phytosterols and oxyphytosterols are less well-known. Over the past few years, different types of foods have been enriched with phytosterols on the basis of the properties of these compounds that reduce circulating cholesterol levels in certain experimental conditions. It is therefore important to gain better knowledge of the risks and benefits of this type of diet. In this study, conducted in human monocytic U937 cells, the ability of phytosterols (sitosterol, campesterol) and oxyphytosterols (7 beta-hydroxysitosterol, 7-ketositosterol) to induce cell death, polar lipid accumulation, and pro-inflammatory cytokine (MCP-1; IL-8) secretion was determined and compared to that of oxysterols (7-ketocholesterol, 7 beta-hydroxycholesterol). Phytosterols and oxyphytosterols had no significant effects on the parameters studied; only 7 beta-hydroxysitosterol slightly increased cell death, whereas at the concentration used (20 mu g/mL), strong cytotoxic effects were observed with the oxysterols. With sitosterol, campesterol, and 7-ketositosterol, IL-8 secretion was decreased, and with campesterol the intracellular polar lipid level was reduced. The data show that phytosterols and oxyphytosterols have no oxysterol-like side effects, and they rather argue in favor of phytosterols' beneficial effects.

The retina is one of the vertebrate tissues with the highest content in polyunsaturated fatty acids (PUFA). A large proportion of retinal phospholipids, especially those found in photoreceptor membranes, are dipolyunsaturated molecular species. Among them, dipolyunsaturated phosphatidylcholine (PC) molecular species are known to contain very-long-chain polyunsaturated fatty acids (VLC-PUFA) from the n-3 and n-6 series having 24-36 carbon atoms (C24-C36) and four to six double bonds. Recent interest in the role played by VLC-PUFA arose from the findings that a protein called elongation of very-long-chain fatty acids 4 (ELOVL4) is involved in their biosynthesis and that mutations in the ELOVL4 gene are associated with Stargardt-like macular dystrophy (STD3), a dominantly inherited juvenile macular degeneration leading to vision loss. The aim of the present study was to develop an HPLC-ESI-MS/MS method for the structural characterisation and the quantification of dipolyunsaturated PC molecular species containing VLC-PUFA and validate this methodology on retinas from bovines and human donors. Successful separation of phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), PC, lyso-phosphatidylcholine (LPC) and sphingomyelin (SM) was achieved using a silica gel column and a gradient of hexane/isopropanol/water containing ammonium formate as a mobile phase. A complete structural characterisation of intact phosphatidylcholine species was obtained by collision-induced dissociation (CID) in the negative mode. Fatty acid composition and distribution can be clearly assigned based on the intensity of sn-2/sn-1 fragment ions. The PC species were characterised on bovine retina, 28 of which were dipolyunsaturated PC species containing one VLC-PUFA (C24-C36) with three to six double bonds. VLC-PUFA was always in the sn-1 position while PUFA at the sn-2 position was exclusively docosahexaenoic acid (DHA, C22:6n-3). Most of these VLC-PUFA-containing dipolyunsaturated PCs were detected and quantified in human retinas. The quantitative analysis of the different PC molecular species was performed in the positive mode using precursor ion scanning of m/z 184 and 14:0/14:0-PC and 24:0/24:0-PC as internal standards. The relationship between the MS peak intensities of different PC species and their carbon chain length was included for calibration. The main compounds represented were those having VLC-PUFA with 32 carbon atoms (C32:3, C32:4, C32:5 and C32:6) and 34 carbon atoms (C34:3, C34:4, C34:5 and C34:6). Dipolyunsaturated PCs with 36:5 and 36:6 were detected but in smaller quantities. In conclusion, this new HPLC-ESI-MS/MS method is sensitive and specific enough to structurally characterise and quantify all molecular PC species, including those esterified with VLC-PUFA. This technique is valuable for a precise characterisation of PC molecular species containing VLC-PUFA in retina and may be useful for a better understanding of the pathogenesis of STD3.

Purpose: To estimate the association and interaction of single nucleotide polymorphism (SNP) in cholesterol-24S-hydroxylase (CYP46A1) gene with HTRA1, LOC387715, CFH SNPs with age-related macular degeneration (AMD) in a North-East French population. Methods: Cross-sectional study involving 142 AMD patients with exudative AMD or geographic atrophy and 70 unrelated control subjects. SNPs were genotyped in CYP46A1, HTRA1, LOC387715, and CFH genes. Plasma 24S-hydroxycholesterol, the metabolic product of CYP46A1, was quantified. Sex, age, alleles, and genotype frequencies between AMD patients and controls were compared using the ² and Student t-tests. Odd-ratios (OR) and 95% confidence intervals (CI) were calculated by logistic regression to assess the relative association between disease and age, sex, and genotypes. Results: The SNP rs754203 in CYP46A1 was not associated with AMD (OR=1.1, 95% CI=0.78-1.43, p=0.76). The OR for risk of AMD was 2.1 (95% CI=1.1-4.4, p=0.03) for the A-allele of rs11200638 in HTRA1, 2.8 (95% CI=1.4-5.5, p=0.002) for the T-allele of rs10490924 in LOC387715, and 1.5 (95% CI=1.1-2.0, p=0.004) for the C-allele for rs1061170 in CFH. These associations were found only in patients with exudative AMD but not with geographic atrophy. An OR of 11.3 (95% CI=0.7-170, p=0.003) was obtained for carriers with both CC-genotype in CFH and TT in CYP46A1. Conclusions: The TT-genotype of rs754203 in CYP46A1 conferred a higher risk for exudative AMD in patients who carry the CC-genotype of rs1061170 in CFH.

Purpose Accumulation of lipids, and especially of cholesteryl esters, under the retinal pigment epithelium and within Bruch’s membrane is a normal feature of aging and has also been observed in human eyes with age-related maculopathy. Our objective was to evaluate the retinal phenotype of apoB100,LDLR-/- mice, a model for lipid metabolism dysfunction and potentially of aging of the retina. Methods ApoB100,LDLR-/- mice were studied at 7 and 14 months of age by standard scotopic and photopic electroretinography by comparison to control animals. Fundus images were obtained with a confocal SLO (Heidelberg Retina Angiograph). The integrity of the vascular system was investigated by means of fluoresceine and indocyanine green angiography. Sections of eye cups were stained by filipin to detect cholesterol deposits. Results Both scotopic and photopic b-wave amplitudes were reduced in apoB100,LDLR-/- mice compared to control mice (Rmax=125 µV vs 208 µV for the scotopic b-wave amplitude at 7 months, and 83 µV vs 162 µV at 14 months). Similarly rods and cones sensitivity was 0.5log unit lower in apoB100,LDLR-/- mice at 14 months, compared to control mice. Although the retinal and the choroidal vascular systems were normal, apoB100,LDLR-/- mice displayed white auto-fluorescent dots in the retinal pigment epithelium layer which likely corresponded to cholesterol deposits. Conclusion The present apoB100,LDLR-/- mouse, is one of the only models with neutral lipid deposits at the basement of RPE that can potentially be very useful to study the mechanisms of lipid deposition that occurs universally in human retina while aging.

Purpose. Genetics has made significant contributions to the study of glaucoma over the past few decades. Cholesterol-24S-hydroxylase (CYP46A1) is a cholesterol-metabolizing enzyme that is especially expressed in retinal ganglion cells. CYP46A1 and its metabolic product, 24S-hydroxycholesterol, have been linked to neurodegeneration. A single-nucleotide polymorphism (SNP) in the CYP46A1 gene, designated as rs754203 and associated with Alzheimer disease, was evaluated as a genetic risk factor for primary open-angle glaucoma (POAG), as well as plasma 24S-hydroxycholesterol levels. Methods. The frequency of the CYP46*C and CYP46*T alleles was analyzed in 150 patients with POAG and 118 control subjects. Plasma 24S-hydroxycholesterol levels were quantified. Sex, age, alleles, and genotype frequencies between patients with POAG and control subjects were compared by using the {chi}2 and Student's t-tests. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by logistic regression to assess the relative association between disease and age, sex, and genotypes. Results. The frequency of the TT genotype was significantly higher in patients with POAG than in control subjects (61.3% versus 48.3%, respectively, OR = 1.26; 95% CI = 1.006–1.574, P < 0.05). Plasma 24S-hydroxycholesterol levels did not differ between control subjects and patients with POAG. The ratio of estimated brain weight to liver volume as an estimate of the capacity of the human body to synthesize and metabolize 24S-hydroxycholesterol was found to correlate to plasma 24S-hydroxycholesterol in control subjects and patients with POAG. Conclusions. The rs754203 SNP in CYP46A1 was associated with a risk for POAG. This polymorphism was not associated with changes in plasma 24S-hydroxycholesterol, highlighting that despite its retinal origin, 24S-hydroxycholesterol cannot be used as a biomarker for POAG.