Polyunsaturated Fatty Acids Arachidonic Acid Research Articles

Omega-6 and Omega-3 Polyunsaturated Fatty Acids and Allergic Diseases in Infancy and Childhood

There may be a causal relationship between intake of n-6 polyunsaturated fatty acids (PUFAs) and childhood allergic diseases. This can be explained by plausible biological mechanisms involving eicosanoid mediators produced from the n-6 PUFA arachidonic acid. Long chain n-3 PUFAs are found in fish and fish oils. These fatty acids act to oppose the actions of n-6 PUFAs. Thus, it is considered that n-3 PUFAs will lower the risk of developing allergic diseases. In support of this, protective associations have been reported between maternal fish intake during pregnancy and allergic outcomes in infants and children from those pregnancies. However, studies of fish intake during infancy and childhood and allergic outcomes in those infants or children are inconsistent, although some reported a protective association. Supplementing pregnant women with fish oil can induce immunologic changes in cord blood. This supplementation has been reported in some studies to decrease sensitisation to common food allergens and to lower the prevalence and severity of atopic dermatitis in the first year of life. The protective effect of maternal n-3 PUFAs may last until adolescence of the offspring. Fish oil supplementation in infancy may decrease the risk of developing some manifestations of allergic disease, although this benefit may not persist. Whether fish oil is a useful therapy in children with asthma receiving standard therapy is not clear from studies performed to date and this requires further exploration.

Newborns with lower levels of circulating polyunsaturated fatty acids (PUFA) are abdominally more adipose

Maternal nutrition is the main source of Poly-Unsaturated Fatty Acids (PUFA) for the fetus. PUFA may influence the accumulation of fat in early life. In 33 breastfed infants born appropriate-for-gestational-age, we studied whether body composition (judged by absorptiometry at 2 wk and 4 mo) relates to PUFA levels (assessed by gas chromatography) in the maternal or fetal circulation at birth. Abdominal fat at 2 wk associated negatively to umbilical-cord levels of separate PUFA (linoleic, arachidonic, eicosapentanoic and docosahexaenoic acid; all P between 0.001 and 0.015). Collectively, the assessed n-6 PUFA on one hand and the n-3 PUFA on the other hand associated negatively to the absolute amount of abdominal fat (in grams; P = 0.001 and P = 0.002, respectively) and to the relative amount of abdominal fat (fraction of total fat; P = 0.001 and P = 0.006, respectively). No other significant associations were observed. In conclusion, newborns with lower levels of circulating PUFA were found to be abdominally more adipose. The mechanisms underpinning these associations remain to be determined.

Polyunsaturated fatty acid associations with dopaminergic indices in major depressive disorder

Dopaminergic function is thought to be altered in major depression and, in animal studies, is reduced in omega-3 polyunsaturated fatty acid (PUFA) deficiency states. Therefore we studied PUFAs and resting prolactin, a marker for dopaminergic tone, and cerebrospinal fluid homovanillic acid (HVA), the chief dopamine metabolite. In medication-free adults (n = 23) with DSM-IV major depressive disorder (MDD), we measured plasma phospholipid levels of omega-3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the omega-6 PUFA arachidonic acid (AA), and plasma prolactin levels before and after administration of dl-fenfluramine (FEN). In a subset of patients (n = 14), cerebrospinal fluid levels of HVA and the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were obtained through lumbar puncture. Baseline prolactin was negatively correlated with omega-3 PUFAs (logDHA, F(1,21) = 20.380, p < 0.001; logEPA, F(1,21) = 10.051, p = 0.005) and positively correlated with logAA:DHA (F(1,21) = 15.263, p = 0.001), a measure of omega-6/omega-3 balance. LogDHA was negatively correlated with CSF HVA (Spearman's ρ = -0.675, p = 0.008) but not 5-HIAA (Spearman's ρ = -0.143, p = 0.626) after controlling for sex and HVA - 5-HIAA correlation. PUFAs did not predict the magnitude of the FEN-stimulated change in prolactin, considered to be a serotonin effect. The robust relationship of omega-3 PUFAs with dopaminergic but not serotonergic indices suggests that omega-6:omega-3 balance may impact depression pathophysiology through effects on the dopaminergic system.

Association between serum omega-3 to omega-6 polyunsaturated fatty acid ratio and cardiovascular events in a general Japanese population

Inflammation is recognized to play an important role in the development of atherosclerotic diseases, such as coronary heart disease (CHD). Polyunsaturated fatty acids (PUFAs) are important constituents of cell membrane phospholipids, and the phospholipids of inflammatory cells are rich in omega-6 PUFA arachidonic acid (AA), with low amounts of omega-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) [1]. However, AA acts as a source of pro-inflammatory eicosanoids, whereas both EPA and DHA reduce AA-derived eicosanoids and pro-inflammatory cytokines and produce anti-inflammatory resolvins and protectins [1–3].

Changes in plasma and erythrocyte omega-6 and omega-3 fatty acids in response to intravenous supply of omega-3 fatty acids in patients with hepatic colorectal metastases

BackgroundEicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are functionally the most important omega-3 polyunsaturated fatty acids (PUFAs). Oral supply of these fatty acids increases their levels in plasma and cell membranes, often at the expense of the omega-6 PUFAs arachidonic acid (ARA) and linoleic acid. This results in an altered pattern of lipid mediator production to one which is less pro-inflammatory. We investigated whether short term intravenous supply of omega-3 PUFAs could change the levels of EPA, DHA, ARA and linoleic acid in plasma and erythrocytes in patients with hepatic colorectal metastases.MethodsTwenty patients were randomised to receive a 72 hour infusion of total parenteral nutrition with (treatment group) or without (control group) omega-3 PUFAs. EPA, DHA, ARA and linoleic acid were measured in plasma phosphatidylcholine (PC) and erythrocytes at several times points up to the end of infusion and 5 to 12 days (mean 9 days) after stopping the infusion.ResultsThe treatment group showed increases in plasma PC EPA and DHA and erythrocyte EPA and decreases in plasma PC and erythrocyte linoleic acid, with effects most evident late in the infusion period. Plasma PC and erythrocyte EPA and linoleic acid all returned to baseline levels after the 5–12 day washout. Plasma PC DHA remained elevated above baseline after washout.ConclusionsIntravenous supply of omega-3 PUFAs results in a rapid increase of EPA and DHA in plasma PC and of EPA in erythrocytes. These findings suggest that infusion of omega-3 PUFAs could be used to induce a rapid effect especially in targeting inflammation.Trial registrationhttp://www.clinicaltrials.gov identifier NCT00942292

Ligand-independent activation of EphA2 by arachidonic acid induces metastasis-like behaviour in prostate cancer cells

Background:High intake of omega-6 polyunsaturated fatty acids (PUFA) has been associated with clinical progression in prostate cancer (CaP). This study investigates the signalling mechanism by which the omega-6 PUFA arachidonic acid (AA) induces prostatic cellular migration to bone marrow stroma.Methods:Western blot analysis of the PC-3, PC3-GFP, DU 145 and LNCaP cells or their lipid raft (LR) components post AA stimulation was conducted in association with assays for adhesion and invasion through the bone marrow endothelial monolayers.Results:Arachidonic acid increased transendothelial migration of PC3-GFP cells (adhesion 37%±0.08, P=0.0124; transmigration 270%±0.145, P=0.0008). Akt, Src and focal adhesion kinase (FAK) pathways were induced by AA and integrally involved in transendothelial migration. LR were critical in AA uptake and induced Akt activity. Ephrin receptor A2 (EphA2), localised in LR, is expressed in DU 145 and PC-3 cells. Arachidonic acid induced a rapid increase of EphA2 Akt-dependent/ligand-independent activation, while knockdown of the EphrinA1 ligand decreased AA induced transendothelial migration, with an associated decrease in Src and FAK activity. Arachidonic acid activated Akt in EphA2− LNCaP cells but failed to induce BMEC transendothelial invasion.Conclusion:Arachidonic acid induced stimulation of EphA2 in vitro is associated fundamentally with CaP epithelial migration across the endothelial barrier.

Improved Mitochondrial Function with Diet-Induced Increase in Either Docosahexaenoic Acid or Arachidonic Acid in Membrane Phospholipids

Mitochondria can depolarize and trigger cell death through the opening of the mitochondrial permeability transition pore (MPTP). We recently showed that an increase in the long chain n3 polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA; 22:6n3) and depletion of the n6 PUFA arachidonic acid (ARA; 20:4n6) in mitochondrial membranes is associated with a greater Ca2+ load required to induce MPTP opening. Here we manipulated mitochondrial phospholipid composition by supplementing the diet with DHA, ARA or combined DHA+ARA in rats for 10 weeks. There were no effects on cardiac function, or respiration of isolated mitochondria. Analysis of mitochondrial phospholipids showed DHA supplementation increased DHA and displaced ARA in mitochondrial membranes, while supplementation with ARA or DHA+ARA increased ARA and depleted linoleic acid (18:2n6). Phospholipid analysis revealed a similar pattern, particularly in cardiolipin. Tetralinoleoyl cardiolipin was depleted by 80% with ARA or DHA+ARA supplementation, with linoleic acid side chains replaced by ARA. Both the DHA and ARA groups had delayed Ca2+-induced MPTP opening, but the DHA+ARA group was similar to the control diet. In conclusion, alterations in mitochondria membrane phospholipid fatty acid composition caused by dietary DHA or ARA was associated with a greater cumulative Ca2+ load required to induced MPTP opening. Further, high levels of tetralinoleoyl cardiolipin were not essential for normal mitochondrial function if replaced with very-long chain n3 or n6 PUFAs.

A comparative study of tissue ω-6 and ω-3 polyunsaturated fatty acids (PUFA) in benign and malignant pathologic stage pT2a radical prostatectomy specimens

ObjectiveTo analyze different polyunsaturated fatty acid (PUFA) tissue levels in malignant compared with benign prostatic tissue from the same prostate specimens. Materials and methodsFresh frozen benign and malignant prostatic tissue was obtained from radical prostatectomy specimens in 49 men with pathologic stage pT2a prostate cancer. Histopathologic examination confirmed that all tissues from each prostate being analyzed were either completely benign or almost totally malignant. The PUFA composition in these tissues was determined by gas-liquid chromatography on a capillary column. The relative amount of each PUFA (% of total fatty acids) was quantified by integrating the area under the peak and dividing the result by the total area of all fatty acids. ResultsTissue levels of dihomo-γ-linolenic acid, (C20:3w6), an ω-6 PUFA and a major precursor of ω-6 PUFA metabolites, were significantly higher in malignant compared with benign tissues (P = 0.002). Tissue levels of the downstream ω-6 metabolites, arachidonic acid (AA) (20:4ω6), and adrenic acid, (22:4ω6), were significantly lower in cancer tissues, (P < 0.0001 and P = 0.013, respectively). Overall, the total levels of ω-6 PUFA were lower in cancer (P = 0.001). ConclusionWe found that the ω-6 PUFA AA and adrenic acid are decreased in malignant prostatic tissues compared with benign tissues from the same prostates. These findings provide additional evidence that dietary fat is associated with prostatic carcinogenesis.

Validation of a Food Frequency Questionnaire to Assess Intake of n-3 Polyunsaturated Fatty Acids in Subjects with and without Major Depressive Disorder

The role of n-3 polyunsaturated fatty acids (PUFAs) in psychiatric illness is a topic of public health importance. This report describes development and biomarker validation of a 21-item, self-report food frequency questionnaire (FFQ) intended for use in psychiatric research to assess intake of α-linolenic acid (18:3n-3 [ALA]), docosahexaenoic acid (22:6n-3 [DHA]), and eicosapentaenoic acid (20:5n-3 [EPA]). In a cross-sectional study conducted from September 2006 to September 2008, sixty-one ethnically diverse adult participants with (n=34) and without (n=27) major depressive disorder completed this n-3 PUFA FFQ and provided a plasma sample. Plasma levels of n-3 PUFAs EPA and DHA, and n-6 PUFA arachidonic acid (20:4n-6 [AA]) were quantified by gas chromatography. Using Spearman's ρ, FFQ-estimated intake correlated with plasma levels of DHA ( r=0.50; P<0.0001) and EPA ( r=0.38; P=0.002), but not with ALA levels ( r=0.22; P=0.086). Participants were classified into quartiles by FFQ-estimated intake and plasma PUFA concentrations. Efficacy of the FFQ to rank individuals into same or adjacent plasma quartiles was 83% for DHA, 78.1% for EPA, and 70.6% for ALA; misclassification into extreme quartiles was 4.9% for DHA, 6.5% for EPA, and 8.2% for ALA. FFQ-estimated EPA intake and plasma EPA were superior to plasma AA levels as predictors of the plasma AA to EPA ratio. This brief FFQ can provide researchers and clinicians with valuable information concerning dietary intake of DHA and EPA.

Ethyl-eicosapentaenoate modulates changes in neurochemistry and brain lipids induced by parkinsonian neurotoxin 1-methyl-4-phenylpyridinium in mouse brain slices

Evidence suggests a link between Parkinson's disease and the dietary intake of omega (n)−3 and n−6 polyunsaturated fatty acids (PUFAs). Presently, we investigated whether an acute dose of parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP +) affects brain n−3 and n−6 PUFA content and expression of fatty acid metabolic enzymes cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2) in brain slices from C57Bl/6 mice. Furthermore, we investigated whether feeding a diet of n−3 PUFA ethyl-eicosapentaenoate (E-EPA) to these mice can attenuate the MPP + induced changes in brain PUFA content and expression of cPLA2 and COX-2, and attenuate MPP + induced changes in neurotransmitters and metabolites and apoptotic markers, bax, bcl-2 and caspase-3. MPP + increased brain content of n−6 PUFAs linoleic acid and arachidonic acid, and increased the mRNA expression of cPLA2. MPP + also depleted striatal dopamine levels and increased dopamine turnover, and depleted noradrenaline levels in the frontal cortex. The neurotoxin induced increases in bax, bcl-2 and caspase-3 mRNA expression that approached significance. E-EPA by itself increased brain n−3 content, including EPA and docosapentaenoic acid (C22:5, n−3), and increased cortical dopamine. More importantly, E-EPA attenuated the MPP + induced increase in n−6 fatty acids content, partially attenuated the striatal dopaminergic turnover, and prevented the increases of pro-apoptotic bax and caspase-3 mRNAs. In conclusion, increases in n−6 PUFAs in the acute stage of exposure to parkinsonian neurotoxins may promote pro-inflammatory conditions. EPA may provide modest beneficial effects in Parkinson's disease, but further investigation is warranted.

Diversity of caecal bacteria is altered in interleukin-10 gene-deficient mice before and after colitis onset and when fed polyunsaturated fatty acids

Interleukin-10 gene-deficient (Il10(-/-)) mice show a hyper-reaction to normal intestinal bacteria and develop spontaneous colitis similar to that of human Crohn's disease when raised under conventional (but not germ-free) conditions. The lack of IL10 protein in these mice leads to changes in intestinal metabolic and signalling processes. The first aim of this study was to identify changes in the bacterial community of the caeca at 7 weeks of age (preclinical colitis) and at 12 weeks of age (when clinical signs of colitis are present), and establish if there were any changes that could be associated with the mouse genotype. We have previously shown that dietary n-3 and n-6 polyunsaturated fatty acids (PUFA) have anti-inflammatory effects and affect colonic gene expression profiles in Il10(-/-) mice; therefore, we also aimed to test the effect of the n-3 PUFA eicosapentaenoic acid (EPA) and the n-6 PUFA arachidonic acid (AA) on the bacterial community of caeca in both Il10(-/-) and C57 mice fed these diets. The lower number of caecal bacteria observed before colitis (7 weeks of age) in Il10(-/-) compared to C57 mice suggests differences in the intestinal bacteria that might be associated with the genotype, and this could contribute to the development of colitis in this mouse model. The number and diversity of caecal bacteria increased after the onset of colitis (12 weeks of age). The increase in caecal Escherichia coli numbers in both inflamed Il10(-/-) and healthy C57 mice might be attributed to the dietary PUFA (especially dietary AA), and thus not be a cause of colitis development. A possible protective effect of E. coli mediated by PUFA supplementation and associated changes in the bacterial environment could be a subject for further investigation to define the mode of action of PUFA in colitis.

Atopy Risk in Infants and Children in Relation to Early Exposure to Fish, Oily Fish, or Long-Chain Omega-3 Fatty Acids: A Systematic Review

There are two main families of polyunsaturated fatty acids (PUFAs), the n-6 and the n-3 families. It has been suggested that there is a causal relationship between n-6 PUFA intake and allergic disease, and there are biologically plausible mechanisms, involving eicosanoid mediators of the n-6 PUFA arachidonic acid, that could explain this. Fish and fish oils are sources of long-chain n-3 PUFAs and these fatty acids act to oppose the actions of n-6 PUFAs. Thus, it is considered that n-3 PUFAs will protect against atopic sensitization and against the clinical manifestations of atopy. Evidence to examine this has been acquired from epidemiologic studies investigating associations between fish intake in pregnancy, lactation, infancy, and childhood, and atopic outcomes in infants and children and from intervention studies with fish oil supplements in pregnancy, lactation, infancy, and childhood, and atopic outcomes in infants and children. All five epidemiological studies investigating the effect of maternal fish intake during pregnancy on atopic or allergic outcomes in infants/children of those pregnancies concluded protective associations. One study investigating the effects of maternal fish intake during lactation did not observe any significant associations. The evidence from epidemiological studies investigating the effects of fish intake during infancy and childhood on atopic outcomes in those infants or children is inconsistent, although the majority of the studies (nine of 14) showed a protective effect of fish intake during infancy or childhood on atopic outcomes in those infants/children. Fish oil supplementation during pregnancy and lactation or during infancy or childhood results in a higher n-3 PUFA status in the infants or children. Fish oil provision to pregnant women is associated with immunologic changes in cord blood and such changes may persist. Studies performed to date indicate that provision of fish oil during pregnancy may reduce sensitization to common food allergens and reduce prevalence and severity of atopic dermatitis in the first year of life, with a possible persistence until adolescence with a reduction in eczema, hay fever, and asthma. Fish oil provision to infants or children may be associated with immunologic changes in the blood but it is not clear if these are of clinical significance and whether they persist. Fish oil supplementation in infancy may decrease the risk of developing some manifestations of allergic disease, but this benefit may not persist as other factors come into play. It is not clear whether fish oil can be used to treat children with asthma as the two studies conducted to date give divergent results. Further studies of increased long-chain n-3 PUFA provision in during pregnancy, lactation, and infancy are needed to more clearly identify the immunologic and clinical effects in infants and children and to identify protective and therapeutic effects and their persistence.

Influence of omega-6 PUFA arachidonic acid and bone marrow adipocytes on metastatic spread from prostate cancer

Background:Prostate cancer (CaP) preferentially metastasises to the bone, and we have previously shown that the poly-unsaturated fatty acid (PUFA) arachidonic acid (AA) is a potent stimulator of CaP invasion. Here we present that AA promotes CaP invasion by inducing bone marrow adipocyte formation.Methods:Boyden invasion-chamber assays assessed the ability of dietary oils, their PUFA components, and specific PUFA-loaded adipocytes to induce PC-3 invasion. Lipid transfer and metabolism was followed using deuterated AA and Fourier Transform Infrared spectroscopy (FTIR).Results:Poly-unsaturated fatty acid constituents, but not their corresponding dietary oils, induced PC-3 invasion. PUFAs induce bone marrow adipocyte (BM-Ad) differentiation with AA inducing higher levels of BM-Ad differentiation, as compared with other PUFAs (3998±514.4 vs 932±265.8; P=0.00002), which stimulated greater PC-3 invasion than free AA (22 408.5±607.4 vs 16 236±313.9; P=0.01111) or adipocytes generated in the presence of other PUFAs. In bone marrow co-culture PC-3 and BM-Ad interactions result in direct uptake and metabolism of AA by PC-3 cells, destruction of the adipocyte and subsequent formation of a bone metastasis.Conclusion:The data supports the hypothesis that AA not only promotes CaP invasion, it also prepares the ‘soil’, making it more supportive for implantation and propagation of the migrating metastatic cell.

Polyunsaturated Fatty Acids and Inflammation: Therapeutic Potential in Rheumatoid Arthritis

The fatty acids of most relevance to inflammatory processes are the n-6 polyunsaturated fatty acid (PUFA) arachidonic acid, the precursor of inflammatory eicosanoids like prostaglandin E2 and leukotriene B4, and the n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). EPA and DHA are found in oily fish and fish oils. Eicosanoids derived from the n-6 PUFA arachidonic acid play a role in rheumatoid arthritis (RA), and the efficacy of nonsteroidal antiinflammatory drugs in RA indicates the importance of pro-inflammatory cyclooxygenase pathway products of arachidonic acid in the pathophysiology of the disease. EPA and DHA inhibit arachidonic acid metabolism to inflammatory eicosanoids. EPA gives rise to eicosanoid mediators that are less inflammatory than those produced from arachidonic acid and both EPA and DHA give rise to resolvins that are anti-inflammatory and inflammation resolving. N- 3 PUFAs exert effects on other aspects of immunity relevant to RA like leukocyte chemotaxis, antigen presentation, T cell reactivity and inflammatory cytokine production. Fish oil has been shown to slow the development of arthritis in an animal model and to reduce disease severity. Randomised clinical trials have demonstrated a range of clinical benefits of fish oil in patients with RA including reducing pain, duration of morning stiffness and use of non-steroidal antiinflammatory drugs.

Effect of Dietary Fatty Acids on Inflammatory Gene Expression in Healthy Humans

Over the past 100 years, changes in the food supply in Western nations have resulted in alterations in dietary fatty acid consumption, leading to a dramatic increase in the ratio of omega-6 (omega6) to omega3 polyunsaturated fatty acids (PUFA) in circulation and in tissues. Increased omega6/omega3 ratios are hypothesized to increase inflammatory mediator production, leading to higher incidence of inflammatory diseases, and may impact inflammatory gene expression. To determine the effect of reducing the omega6/omega3 ratio on expression of inflammatory pathway genes in mononuclear cells, healthy humans were placed on a controlled diet for 1 week, then given fish oil and borage oil for an additional 4 weeks. Serum and neutrophil fatty acid composition and ex vivo leukotriene B(4) production from stimulated neutrophils were measured at the start and end of the supplementation period and after a 2-week washout. RNA was isolated from mononuclear cells and expression of PI3K, Akt, NFkappaB, and inflammatory cytokines was measured by real-time PCR. A marked increase was seen in serum and neutrophil levels of long-chain omega3 PUFA concomitant with a reduction in the omega6/omega3 PUFA ratio (40%). The ex vivo capacity of stimulated neutrophils to produce leukotriene B(4) was decreased by 31%. Expression of PI3Kalpha and PI3Kgamma and the quantity of PI3Kalpha protein in mononuclear cells was reduced after supplementation, as was the expression of several proinflammatory cytokines. These data reveal that PUFA may exert their clinical effects via their capacity to regulate the expression of signal transduction genes and genes for proinflammatory cytokines.

S2025 Negative Predictive Value of PET/CT in a Large Series of Patients with a Clinical Suspicion of Pancreatic Cancer

Introduction: Epidemiologic and experimental studies have shown that dietary intake of n3 polyunsaturated fatty acid (PUFA) reduces cancer risk. We have previously reported that in contrast to the n-6 PUFA arachidonic acid (AA) the n-3 PUFA eicosapentaenoic acid (EPA) decreased pancreatic cancer (PaCa) growth. However, the observed effects using standard two dimensional (2D) culture conditions were only modest. It has become increasingly recognized that the phenotype of cultured cancer cells changes if cells are allowed to grow in more physiologically relevant three-dimensional (3D) assays. The aim of the present study was therefore to assess the effects of AA and EPA on PaCa cell growth using 3D growth assays. Methods and results: The human PaCa cell lines MIA PaCa-2 with (MP2+COX2) or without (MP2-COX2) stable COX-2 expression, and BxPC-3, which express wildtype COX2, were used for our studies. Culture dishes were coated with 50% Matrigel (diluted 1:1 with serum-free culture medium). Cells were seeded on top and overlaid with serum-free culture medium. Fatty acids were present in the Matrigel and the culture medium on top. Colony formation and cell growth were measured after 5-7 days by counting and BrdU incorporation. For comparison, standard 2D growth was evaluated using MTT assays. Exposure of MP2+COX2 cells to AA (5 μM) in the 3D assay increased the growth by more than 10-fold (count) and 2.8-fold (BrdU incorporation), while it enhanced growth only by 1.5fold in the 2D assay (MTT). EPA (200 μM) decreased growth in 3D assays by more than 10-fold but only by 1.6-fold in 2D assays. Similar effects were seen in the COX-2 expressing BxPC-3 cells. In MP2-COX2 cells, AA had only a marginal growth-enhancing effect, which was still more pronounced in 3D assays (2.5-fold increase in cell count vs. 1.3-fold increase in MTT). EPA also markedly decreased cell growth in MP2-COX2 cells with a more than 10-fold reduction in 3D assays but only a 3.6-fold decrease in 2D assays. Conclusion: In pancreatic cancer cells the growth enhancing effects of the n-6 PUFA AA as well as the growth inhibitory effects of the n-3 PUFA EPA were significantly more pronounced in 3D culture assays compared to standard 2D assays. AA significantly increased the growth of COX-2 positive pancreatic cancer cells, while EPA decreased the growth of COX-2 positive and -negative pancreatic cancer cells. This suggests that the effects of EPA on pancreatic cancer cell growth are at least in part mediated by COX-2 independent mechanisms. Our data suggest that the physiologically more relevant 3D culture system should be the preferred model to evaluate drug efficiency In Vitro.

S2022 A Most Valuable Immunohistochemical Panel for Intestinal and Pancreatobiliary Classification in Carcinoma of the Ampulla of Vater

Introduction: Epidemiologic and experimental studies have shown that dietary intake of n3 polyunsaturated fatty acid (PUFA) reduces cancer risk. We have previously reported that in contrast to the n-6 PUFA arachidonic acid (AA) the n-3 PUFA eicosapentaenoic acid (EPA) decreased pancreatic cancer (PaCa) growth. However, the observed effects using standard two dimensional (2D) culture conditions were only modest. It has become increasingly recognized that the phenotype of cultured cancer cells changes if cells are allowed to grow in more physiologically relevant three-dimensional (3D) assays. The aim of the present study was therefore to assess the effects of AA and EPA on PaCa cell growth using 3D growth assays. Methods and results: The human PaCa cell lines MIA PaCa-2 with (MP2+COX2) or without (MP2-COX2) stable COX-2 expression, and BxPC-3, which express wildtype COX2, were used for our studies. Culture dishes were coated with 50% Matrigel (diluted 1:1 with serum-free culture medium). Cells were seeded on top and overlaid with serum-free culture medium. Fatty acids were present in the Matrigel and the culture medium on top. Colony formation and cell growth were measured after 5-7 days by counting and BrdU incorporation. For comparison, standard 2D growth was evaluated using MTT assays. Exposure of MP2+COX2 cells to AA (5 μM) in the 3D assay increased the growth by more than 10-fold (count) and 2.8-fold (BrdU incorporation), while it enhanced growth only by 1.5fold in the 2D assay (MTT). EPA (200 μM) decreased growth in 3D assays by more than 10-fold but only by 1.6-fold in 2D assays. Similar effects were seen in the COX-2 expressing BxPC-3 cells. In MP2-COX2 cells, AA had only a marginal growth-enhancing effect, which was still more pronounced in 3D assays (2.5-fold increase in cell count vs. 1.3-fold increase in MTT). EPA also markedly decreased cell growth in MP2-COX2 cells with a more than 10-fold reduction in 3D assays but only a 3.6-fold decrease in 2D assays. Conclusion: In pancreatic cancer cells the growth enhancing effects of the n-6 PUFA AA as well as the growth inhibitory effects of the n-3 PUFA EPA were significantly more pronounced in 3D culture assays compared to standard 2D assays. AA significantly increased the growth of COX-2 positive pancreatic cancer cells, while EPA decreased the growth of COX-2 positive and -negative pancreatic cancer cells. This suggests that the effects of EPA on pancreatic cancer cell growth are at least in part mediated by COX-2 independent mechanisms. Our data suggest that the physiologically more relevant 3D culture system should be the preferred model to evaluate drug efficiency In Vitro.

S2021 Pronounced Effects of N-3 and N-6 Polyunsaturated Fatty Acids in Pancreatic Cancer Cells Using Three-Dimensional Growth Assays

Introduction: Epidemiologic and experimental studies have shown that dietary intake of n3 polyunsaturated fatty acid (PUFA) reduces cancer risk. We have previously reported that in contrast to the n-6 PUFA arachidonic acid (AA) the n-3 PUFA eicosapentaenoic acid (EPA) decreased pancreatic cancer (PaCa) growth. However, the observed effects using standard two dimensional (2D) culture conditions were only modest. It has become increasingly recognized that the phenotype of cultured cancer cells changes if cells are allowed to grow in more physiologically relevant three-dimensional (3D) assays. The aim of the present study was therefore to assess the effects of AA and EPA on PaCa cell growth using 3D growth assays. Methods and results: The human PaCa cell lines MIA PaCa-2 with (MP2+COX2) or without (MP2-COX2) stable COX-2 expression, and BxPC-3, which express wildtype COX2, were used for our studies. Culture dishes were coated with 50% Matrigel (diluted 1:1 with serum-free culture medium). Cells were seeded on top and overlaid with serum-free culture medium. Fatty acids were present in the Matrigel and the culture medium on top. Colony formation and cell growth were measured after 5-7 days by counting and BrdU incorporation. For comparison, standard 2D growth was evaluated using MTT assays. Exposure of MP2+COX2 cells to AA (5 μM) in the 3D assay increased the growth by more than 10-fold (count) and 2.8-fold (BrdU incorporation), while it enhanced growth only by 1.5fold in the 2D assay (MTT). EPA (200 μM) decreased growth in 3D assays by more than 10-fold but only by 1.6-fold in 2D assays. Similar effects were seen in the COX-2 expressing BxPC-3 cells. In MP2-COX2 cells, AA had only a marginal growth-enhancing effect, which was still more pronounced in 3D assays (2.5-fold increase in cell count vs. 1.3-fold increase in MTT). EPA also markedly decreased cell growth in MP2-COX2 cells with a more than 10-fold reduction in 3D assays but only a 3.6-fold decrease in 2D assays. Conclusion: In pancreatic cancer cells the growth enhancing effects of the n-6 PUFA AA as well as the growth inhibitory effects of the n-3 PUFA EPA were significantly more pronounced in 3D culture assays compared to standard 2D assays. AA significantly increased the growth of COX-2 positive pancreatic cancer cells, while EPA decreased the growth of COX-2 positive and -negative pancreatic cancer cells. This suggests that the effects of EPA on pancreatic cancer cell growth are at least in part mediated by COX-2 independent mechanisms. Our data suggest that the physiologically more relevant 3D culture system should be the preferred model to evaluate drug efficiency In Vitro.

Isoprostanes

The isoprostanes (IsoPs) are a unique series of prostaglandin-like compounds formed in vivo via a nonenzymatic mechanism involving the free radical-initiated peroxidation of arachidonic acid. This article summarizes our current knowledge of these compounds. Herein, a historical account of their discovery and the mechanism of their formation are described. A specific class of IsoPs, the F2-IsoPs, are stable, robust molecules that can be measured as indices of endogenous oxidant stress. The utility of these molecules as biomarkers and methods by which these compounds can be quantified are discussed. In addition to the F2-IsoPs, isoprostanes with other prostane ring structures as well as oxidation products with furan and dioxolane rings can be generated from arachidonic acid. And, in more recent years, isoprostane-like compounds have been shown to be formed from polyunsaturated fatty acids including eicosapentaenoic acid [C20:5, omega-3], docosahexaenoic acid [C22:6, omega-3], and adrenic acid [C22:4, omega-6]. These findings will be summarized as well.

Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified α-linolenic acid in unanesthetized rats

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), long-chain n-3 PUFAs important for brain and heart function, can be obtained from dietary fish products or by liver synthesis from alpha-linolenic acid (alpha-LNA). Their daily human dietary requirements are not clear, and their liver synthesis rates in humans and nonhumans are unknown. We estimated whole-body (presumably liver) synthesis rates in unanesthetized rats by infusing [U-(13)C]alpha-LNA intravenously for 2 h and measuring labeled and unlabeled n-3 PUFA in arterial plasma using negative chemical ionization GC-MS. Newly synthesized esterified [(13)C]DHA, [(13)C]EPA, and [(13)C]docosapentaenoic acid (DPA) appeared in arterial plasma after 60 min of infusion, then their concentrations rose in an S-shaped manner. Esterified concentration x plasma volume data were fit with a sigmoidal equation, whose peak first derivatives provided synthesis rates of unlabeled EPA, DPA, and DHA equal to 8.40, 6.27, and 9.84 mumol/day, respectively. The DHA synthesis rate exceeded the published daily rat brain DHA consumption rate by 30-fold, suggesting that liver synthesis from alpha-LNA could maintain brain DHA homeostasis were DHA absent from the diet. This stable isotope infusion method could be used to quantify whole-body DHA synthesis rates in human subjects.