Lipid biomarkers in surface sediments from an unusual coastal upwelling area from the SW Atlantic Ocean

The coastal upwelling off Cabo Frio (SE Brazilian coast, SEBC) represents an exception to the world`s oceans since the majority of the upwelling areas are located in eastern boundary current systems. Cabo Frio represents an interesting area for investigation due to its tight physical-biological interaction and the importance of the region as a major fishery area in the SEBC. We analyzed a suite of lipid biomarkers to apportion the main sources of organic matter in surface sediments of the continental shelf off Cabo Frio, comparing the area to non-upwelling regions off the SEBC (shelf break off Cabo Frio and continental shelf off Ubatuba). During spring and summer (the upwelling period), diatoms are probably the major sources of polyunsaturated fatty acids (PUFAs) and C-28 sterols in surface sediments from Cabo Frio continental shelf. Sediments sampled in winter showed, in contrast, lower relative abundance of PUFAs and higher stanol/stenol ratio values. In deeper regions off Cabo Frio, elevated concentrations of alkenones, 24-methylcholest-5,22E-dien-3 beta-ol and 24-ethylcholest-5-en-3 beta-ol during the spring may be produced by prymnesiophytes or cryptophytes and cyanobacteria, respectively. In Ubatuba, the C-27 and C-28 sterols are likely derived from omnivorous salps and nanoflagellates. At non-upwelling areas, despite the increase in biomarker concentrations during spring and summer, lower concentrations of PUFAs, phytol and algal sterols than in shelf areas off Cabo Frio suggest the importance of the upwelling system to the rapid transfer of organic carbon to surface sediments. Our results suggest that spatial and temporal variability in organic matter production and deposition merits consideration for constraining the carbon budgets in the coastal region off Cabo Frio. (C) 2008 Elsevier Ltd. All rights reserved.

Adattamento e acclimatazione a diverse temperature di lieviti psicrofili obbligati e facoltativi e di lieviti mesofili. Studio della produzione di acidi grassi polinsaturi omega-3 e omega-6 per via fermentativa.

Adaptation and acclimation to different temperatures of obligate psychrophilic, facultative psychrophilic and mesophilic yeasts. Production of ω-3 and ω-6 polyunsaturated fatty acids by fermentative way. Obligate psychrophilic, facultative psychrophilic and mesophilic yeasts were cultured in a carbon rich medium at different temperatures to investigate if growth parameters, lipid accumulation and fatty acid composition were adaptive and/or acclimatory responses. Acclimation of facultative psychrophiles and mesophiles to lower temperature negatively affected their specific growth rate. Obligate psychrophiles exhibited the highest biomass yield (YX/S), followed by facultative psychrophiles, then by mesophiles. The growth temperature did not influence the YX/S of facultative psychrophiles and mesophiles. Acclimation to lower temperature caused the increase in lipid yield (YL/X) in mesophilic yeasts, but did not affect YL/X in facultative psychrophiles. Similar YL/X were found in both facultative and obligated psychrophiles, suggesting that lipid accumulation is not a distinctive character of adaptation to permanently cold environments. The extent of unsaturation of fatty acids was one major adaptive feature of the yeasts which colonize permanently cold ecosystems. Remarkable amounts of α-linolenic acid were found in obligate psychrophiles at the expenses of linoleic acid, whereas it was generally scarce or absent in all the others strains. Increased unsaturation of fatty acids was also an acclimatory response of mesophiles and facultative psychrophiles to lower temperature. It’s well known that omega-3 polyunsaturated fatty acids (PUFAs) display a variety of beneficial effects on various organ systems and diseases, therefore a process for the microbial production of omega-3 PUFAs would be of great interest. This work sought also to investigate if one of the better psychrophilic yeast, Rhodotorula glacialis DBVPG 4785, stimulated by acclamatory responses, produced omega-3 PUFAs. In fact, the adaptation of psychrophilic yeasts to cold niches is related to the production of higher amounts of lipids and to increased unsaturation degree of fatty acids, presumably to maintain membrane fluidity and functionality at low temperatures. Bioreactor fermentations of Rhodotorula glacialis DBVPG 4785 were carried out at 25, 20, 15, 10, 5, 0, and -3°C in a complex medium with high C:N ratio for 15 days. High biomass production was attained at all the temperatures with a similar biomass/glucose yield (YXS), between 0.40 and 0.45, but the specific growth rate of the strain decreased as the temperature diminished. The coefficients YL/X have been measured between a minimum of 0.50 to a maximum of 0.67, but it was not possible to show a clear effect of temperature. Similarly, the coefficient YL/S ranges from a minimum of 0.22 to a maximum of 0.28: again, it does not appear to be any significant changes due to temperature. Among omega-3 PUFAs, only α-linolenic acid (ALA, 18:3n-3) was found at temperatures below to 0°C, while, it’s remarkable, that the worthy arachidonic acid (C20:4,n-6), stearidonic acid (C20:4,n-3) C22:0 and docosahexaenoic acid (C22:6n-3) were produced only at the late exponential phase and the stationary phase of batch fermentations at 0 and -3°C. The docosahexaenoic acid (DHA) is a beneficial omega-3 PUFA that is usually found in fatty fish and fish oils. The results herein reported improve the knowledge about the responses which enable psychrophilic yeasts to cope with cold and may support exploitation of these strains as a new resource for biotechnological applications.

N-3 fatty acids and cardiovascular prevention

In this study we elucidate the role of polyunsaturated fatty acids (PUFAs) in the prevention of cardiovascular diseases, focusing the attention on their role in the modulation of acyl composition of cell lipids and of gene expression. Regarding this latter mechanism, the effectiveness of PUFAs as activators of two transcriptional factors, SREBPs and PPARs, have been considered. Two different model system have been used: primary cultures of neonatal rat cardiomyocytes and an human hepatoma cell line (HepG2). Cells have been supplemented with different PUFAs at physiological concentration, and special attention has been devoted to the main n-3 PUFAs, EPA and DHA. PUFAs influence on global gene expression in cardiomyocytes has been evaluated using microarray technique. Furthermore, since it is not fully elucidated which transcription factors are involved in this modulation in the heart, expression and activation of the three different PPAR isoforms have been investigated. Hepatocytes have been used as experimental model system in the evaluation of PUFAs effect on SREBP activity. SREBPs are considered the main regulator of cholesterol and triglyceride synthesis, which occur mainly in the liver. In both experimental models the modification of cell lipid fatty acid composition subsequent to PUFAs supplementation has been evaluated, and related to the effects observed at molecular level. The global vision given by the obtained results may be important for addressing new researches and be useful to educators and policy makers in setting recommendations for reaching optimal health through good nutrition.

Harmful algae and their potential impacts on the coastal ecosystem: growth and toxin production dynamics

The main goal of the present thesis was to study some harmful algal species which cause blooms in Italian coastal waters, leading to consequences for human health, coastal ecosystem, fishery and tourism. In particular, in the first part of this thesis the toxicity of Adriatic strains of the raphidophyte Fibrocapsa japonica was investigated. Despite several hypotheses have been proposed for the toxic mechanism of the raphidophytes, especially for the species Chattonella antiqua and C. marina, which have been studied more extensively, just a few studies on the toxic effects of these species for different organisms were reported. Moreover, a careful reading of the literature evidenced as any ichthyotoxic events reported worldwide can be linked to F. japonica blooms. Although recently several studies were performed on F. japonica strains from the USA, Japan, Australia, New Zealand, the Netherlands, Germany, and France in order to characterize their growth and toxicity features, the work reported in this thesis results one of the first investigation on the toxic effects of F. japonica for different organisms, such as bacteria, crustaceans and fish. Mortality effects, together with haemolysis of fish erythrocytes, probably due to the relatively high amount of PUFAs produced by this species, were observed. Mortality for fish, however, was reported only at a high cell density and after a long exposition period (9-10 days); moreover a significant increase of H2O2 obtained in the tanks where sea basses were exposed to F. japonica was also relevant. This result may justify the absence of ichthyotoxic events in the Italian coasts, despite F. japonica blooms detected in these areas were characterized by high cell densities. This work reports also a first complete characterization of the fatty acids produced and extracellularly released by the Adriatic F. japonica, and results were also compared with the fatty acid profile of other strains. The absence of known brevetoxins in F. japonica algal extracts was also highlighted, leading to the hypothesis that the toxicity of F. japonica may be due to a synergic effect of PUFAs and ROS. Another microalgae that was studied in this thesis is the benthic dinoflagellate Ostreopsis cf. ovata. This species was investigated with the aim to investigate the effect of environmental parameters on its growth and toxicity. O. cf. ovata, in fact, shows different blooming periods along the Italian coasts and even the reported toxic effects are variable. The results of this work confirmed the high variability in the growth dynamic and toxin content of several Italian strains which were isolated in recent years along the Adriatic and Tyrrhenian Seas. Moreover, the effects of temperature and salinity on the behaviour of the different isolates are in good agreement with the results obtained from field surveys, which evidence as the environmental parameters are important factors modulating O. cf. ovata proliferation. Another relevant result that was highlighted is the anomaly in the production of palytoxin-like compounds reported by one of the studied isolate, in particular the one isolated in 2008 in Ancona (Adriatic Sea). Only this strain reported the absence of two (ovatoxin-b and –c) of the five ovatoxins so far known in the toxin profile and a different relative abundance of the other toxins. The last aspect that was studied in this thesis regards the toxin biosythesis. In fact, toxins produced (palytoxin-like compounds) or supposed to be produced (brevetoxin-like compounds) by O. cf. ovata and F. japonica, respectively, are polyketides, which are highly oxygenated compounds synthesized by complex enzymes known as polyketide synthase (PKS) enzymes. These enzymes are multi-domain complexes that structurally and functionally resemble the fatty acid synthases (FASs). This work reports the first study of PKS proteins in the dinoflagellates O. cf. ovata, C. monotis and in the raphidophyte F. japonica. For the first time some PKSs were identified in these species, confirming the presence of PKS proteins predicted by the in silico translation of the transcripts found in K. brevis also in other species. The identification of O. cf. ovata PKSs and the localization of the palytoxin-like compounds produced by this dinoflagellate in a similar location (chloroplast) as that observed for other dinoflagellate and cyanobacterial toxins provides some indication that these proteins may be involved in polyketide biosynthesis. However, their potential function as fatty acid synthases cannot be ruled out, as plant fatty acid synthesis also occurs within chloroplasts. This last hypothesis is also supported by the fact that in all the investigated species, and in particular in F. japonica, PKS proteins were present. Therefore, these results provide an important contribution to the study of the polyketides and of the involvement of PKS proteins in the toxin biosynthesis.

Chemopreventive effects of eicosapentaenoic acid free fatty acid in a murine model of colitis-associated colorectal cancer

Inflammatory bowel diseases are associated with increased risk of developing colitis-associated colorectal cancer (CAC). Epidemiological data show that the consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of sporadic colorectal cancer (CRC). Importantly, recent data have shown that eicosapentaenoic acid-free fatty acid (EPA-FFA) reduces polyps formation and growth in models of familial adenomatous polyposis. However, the effects of dietary EPA-FFA are unknown in CAC. We tested the effectiveness of substituting EPA-FFA, for other dietary fats, in preventing inflammation and cancer in the AOM-DSS model of CAC. The AOM-DSS protocols were designed to evaluate the effect of EPA-FFA on both initiation and promotion of carcinogenesis. We found that EPA-FFA diet strongly decreased tumor multiplicity, incidence and maximum tumor size in the promotion and initiation arms. Moreover EPA-FFA, in particular in the initiation arm, led to reduced cell proliferation and nuclear β-catenin expression, whilst it increased apoptosis. In both arms, EPA-FFA treatment led to increased membrane switch from ω-6 to ω-3 PUFAs and a concomitant reduction in PGE2 production. We observed no significant changes in intestinal inflammation between EPA-FFA treated arms and AOM-DSS controls. Importantly, we found that EPA-FFA treatment restored the loss of Notch signaling found in the AOM-DSS control, resulted in the enrichment of Lactobacillus species in the gut microbiota and led to tumor suppressor miR34-a induction. In conclusion, our data suggest that EPA-FFA is an effective chemopreventive agent in CAC.

Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages

The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.

Association of macular pigment density with plasma ω-3 fatty acids: the PIMAVOSA study

To assess the correlation between macular pigment optical density and plasma levels of lutein, zeaxanthin, and fatty acids, especially omega-3 polyunsaturated fatty acids (PUFAs).

The ω3-polyunsaturated fatty acid derivatives AVX001 and AVX002 directly inhibit cytosolic phospholipase A(2) and suppress PGE(2) formation in mesangial cells

ω3-polyunsaturated fatty acids (ω3-PUFAs) are known to exert anti-inflammatory effects in various disease models although their direct targets are only poorly characterized.

Reduced bone breakage and increased bone strength in free range laying hens fed omega-3 polyunsaturated fatty acid supplemented diets

INTRODUCTION The omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are the immediate precursors to a number of important mediators of immunity, inflammation and bone function, with products of omega-6 generally thought to promote inflammation and favour bone resorption. Western diets generally provide a 10 to 20-fold deficit in omega-3 PUFAs compared with omega-6, and this is thought to have contributed to the marked rise in incidence of disorders of modern human societies, such as heart disease, colitis and perhaps osteoporosis. Many of our food production animals, fed on grains rich in omega-6, are also exposed to a dietary deficit in omega-3, with perhaps similar health consequences. Bone fragility due to osteoporotic changes in laying hens is a major economic and welfare problem, with our recent estimates of breakage rates indicating up to 95% of free range hens suffer breaks during lay. METHODS Free range hens housed in full scale commercial systems were provided diets supplemented with omega-3 alpha linolenic acid, and the skeletal benefits were investigated by comparison to standard diets rich in omega-6. RESULTS There was a significant 40-60% reduction in keel bone breakage rate, and a corresponding reduction in breakage severity in the omega-3 supplemented hens. There was significantly greater bone density and bone mineral content, alongside increases in total bone and trabecular volumes. The mechanical properties of the omega-3 supplemented hens were improved, with strength, energy to break and stiffness demonstrating significant increases. Alkaline phosphatase (an osteoblast marker) and tartrate-resistant acid phosphatase (an osteoclast marker) both showed significant increases with the omega-3 diets, indicating enhanced bone turnover. This was corroborated by the significantly lower levels of the mature collagen crosslinks, hydroxylysyl pyridinoline, lysyl pyridinoline and histidinohydroxy-lysinonorleucine, with a corresponding significant shift in the mature:immature crosslink ratio. CONCLUSIONS The improved skeletal health in laying hens corresponds to as many as 68million fewer hens suffering keel fractures in the EU each year. The biomechanical and biochemical evidence suggests that increased bone turnover has enhanced the bone mechanical properties, and that this may suggest potential benefits for human osteoporosis.

Fish or n3-PUFA intake and body composition: a systematic review and meta-analysis.

Obesity is a major public health issue and an important contributor to the global burden of chronic disease and disability. Studies indicate that fish and omega-3 polyunsaturated fatty acids (n3-PUFA) supplements may help prevent cardiovascular and metabolic diseases. However, the effect of fish oil on body composition is still uncertain, so we performed a systematic review of randomized controlled trials and the first meta-analysis on the association between fish or fish oil intake and body composition measures. We found evidence that participants taking fish or fish oil lost 0.59 kg more body weight than controls (95% confidence interval [CI]: -0.96 to -0.21). Treatment groups lost 0.24 kg m(-2) (body mass index) more than controls (-0.40 to -0.08), and 0.49 % more body fat than controls (-0.97 to -0.01). Fish or fish oil reduced waist circumference by 0.81 cm (-1.34 to -0.28) compared with control. There was no difference for fat mass and lean body mass. Further research is needed to confirm or refute our findings and to reveal possible mechanisms by which n3-PUFAs might reduce weight.

Antioxidant supplementation attenuates oxidative stress in patients undergoing coronary artery bypass graft surgery.

Ischemia-reperfusion has been reported to be associated with augmented oxidative stress in the course of surgery, which might be causally involved in the onset of atrial fibrillation (AF), the most common arrhythmia after cardiac surgery. We hypothesized that supplementation of antioxidants and n-3 polyunsaturated fatty acids (n-3 PUFAs) might lower the incidence of AF following coronary artery bypass graft (CABG) surgery. In the present study, by monitoring oxidative stress in the course of CABG surgery, we analyzed the efficacy of vitamins (ascorbic acid and α-tocopherol) and/or n-3 PUFAs (eicosapentaenoic acid and docosahexaenoic acid). Subjects (n = 75) were divided into 4 subgroups: control, vitamins, n-3 PUFAs, and a combination of vitamins and n-3 PUFAs. Fluorescent techniques were used to measure the antioxidative capacity, i.e. ability to inhibit oxidation. Total peroxides, endogenous peroxidase activity, and antibodies against oxidized LDL (oLAb) were used as serum oxidative stress biomarkers. Post-operative increase in oxidative stress was associated with the consumption of antioxidants and a simultaneous onset of AF. This was confirmed through an increased peroxide level and a decreased oLAb titer in control and n-3 PUFAs groups, indicating the binding of antibodies to oxidative modified epitopes. In both subgroups that were supplemented with vitamins, total peroxides decreased, and the maintenance of a constant IgG antibody titer was facilitated. However, treatment with vitamins or n-3 PUFAs was inefficient with respect to AF onset and its duration. We conclude that the administration of vitamins attenuates post-operative oxidative stress in the course of CABG surgery.

Disruption of tumor suppressor gene Hint1 leads to remodeling of the lipid metabolic phenotype of mouse liver

A lipidomic and metabolomic investigation of serum and liver from mice was performed to gain insight into the tumor suppressor gene Hint1. A major reprogramming of lipid homeostasis was found in both serum and liver of Hint1-null (Hint(-/-)) mice, with significant changes in the levels of many lipid molecules, as compared with gender-, age-, and strain-matched WT mice. In the Hint1(-/-) mice, serum total and esterified cholesterol were reduced 2.5-fold, and lysophosphatidylcholines (LPCs) and lysophosphatidic acids were 10-fold elevated in serum, with a corresponding fall in phosphatidylcholines (PCs). In the liver, MUFAs and PUFAs, including arachidonic acid (AA) and its metabolic precursors, were also raised, as was mRNA encoding enzymes involved in AA de novo synthesis. There was also a significant 50% increase in hepatic macrophages in the Hint1(-/-) mice. Several hepatic ceramides and acylcarnitines were decreased in the livers of Hint1(-/-) mice. The changes in serum LPCs and PCs were neither related to hepatic phospholipase A2 activity nor to mRNAs encoding lysophosphatidylcholine acetyltransferases 1-4. The lipidomic phenotype of the Hint1(-/-) mouse revealed decreased inflammatory eicosanoids with elevated proliferative mediators that, combined with decreased ceramide apoptosis signaling molecules, may contribute to the tumor suppressor activity of Hint1.

Fatty acid composition, element concentration and isotope ratios of sea ice algae sampled in Rijpfjorden, Svalbard

The accelerating decrease of Arctic sea ice substantially changes the growth conditions for primary producers, particularly with respect to light. This affects the biochemical composition of sea ice algae, which are an essential high-quality food source for herbivores early in the season. Their high nutritional value is related to their content of polyunsaturated fatty acids (PUFAs), which play an important role for successful maturation, egg production, hatching and nauplii development in grazers. We followed the fatty acid composition of an assemblage of sea ice algae in a high Arctic fjord during spring from the early bloom stage to post bloom. Light conditions proved to be decisive in determining the nutritional quality of sea ice algae, and irradiance was negatively correlated with the relative amount of PUFAs. Algal PUFA content decreased on average by 40 % from April to June, while algal biomass (measured as particulate carbon, C) did not differ. This decrease was even more pronounced when algae were exposed to higher irradiances due to reduced snow cover. The ratio of chlorophyll a (chl a) to C, as well as the level of photoprotective pigments, confirmed a physiological adaptation to higher light levels in algae of poorer nutritional quality. We conclude that high irradiances are detrimental to sea ice algal food quality, and that the biochemical composition of sea ice algae is strongly dependent on growth conditions.

Biometrics, fatty acid composition and stable isotopic ratios of five seabird species from Kongsfjorden, Svalbard

Marine birds are important predators in the marine ecosystem, and dietary studies can give useful information about their feeding ecology, food webs and oceanographic variability. The aim of this study was to increase our understanding of the diet and trophic level of the seabirds breeding in Kongsfjorden, Svalbard. We have used fatty acids and stable isotopes, both of which integrate diet information over space and time, to determine trophic relationships in marine food webs. Fatty acid compositions of muscle from Little auk (Alle alle), Brünnich's guillemot (Uria lomvia), Black-legged kittiwake (Rissa tridactyla), Northern fulmar (Fulmarus glacialis) and Glaucous gull (Larus hyperboreus) were determined and compared with their prey species. Canonical analysis (CA) showed that fatty acid composition differed among the five seabird species. Little auk, Black-legged kittiwake and Northern fulmar had high levels of the Calanus markers 20:1n9 and 22:1, indicating that these seabirds are a part of the Calanus food chain. Brünnich's guillemot differed from the other species with much lower levels of 20:1n9 and 22:1. Brünnich's guillemot is a pursuit diver feeding on fish and amphipods deeper in the water column, below 30 m. Glaucous gull also differed from the other seabird species, with a larger variation in the fatty acid composition indicating a more diverse diet. Trophic level analysis placed Little auk at the lowest trophic level, Brünnich's guillemot and Black-legged kittiwake at intermediate levels and Glaucous gull and Northern fulmar at the highest trophic level.

Reproductive long-term effects, endocrine response and fatty acid profile of rabbit does fed diets supplemented with n-3 fatty acids

The effect of a diet enriched with polyunsaturated n -3 fatty acids (PUFA) on endocrine, reproductive, and productive responses of rabbit females and the litters has been studied. Nulliparous does ( n = 125) were fed ad libitum from rearing to second weaning two diets supplemented with different fat sources: 7.5 g/kg lard for the control diet (group C; n = 63) or 15 g/kg of a commercial supplement containing a 50% ether extract and 35% of total fatty acids (FAs) as PUFA n -3 (Group P; n = 62). Dietary treatments did not affect apparent digestibility coefficients of nutrients, or reproductive variables of does including milk pro- duction, mortality and average daily gain of kits over two lactations. However, on Day 5 and 7 post-induction of ovulation, progesterone of Group P tended to increase to a greater extent than in does of Group C. Total PUFAs, n -6 and n -3 and eicosapentanoic (EPA) contents were greater in adipose tissues of does in Group P than in Group C. Docosapentaenoic acid (DPA), EPA, and docosahexaenoic acid (DHA) concentrations were greater in peri-ovarian than in scapular fat with abdominal fat being intermediate in concentration. In PUFA sup- plemented does, kit mortality at the second parturition tended to be less than in control does. Also, kits born to does of the PUFA-supplemented group weighed more and were of greater length than from does of control group. In conclusion, effectiveness of dietary intervention on reproductive and performance response is greater in the second parity, which suggests an accumulative long-term beneficial effect of n -3 FA supplementation in reproductive rabbit does