High Concentrations Of Arachidonic Acid Research Articles

Oxylipin dynamics in dairy cows during clinical ketosis and after treatment with niacin and flunixin meglumine

Dairy cows with clinical ketosis (CK) exhibit metabolic changes, including intense adipose tissue (AT) lipolysis and systemic insulin resistance that increase plasma BHB and free fatty acids (FFA). Cows with CK also have systemic inflammation, predisposing them to inflammatory and infectious diseases. This inflammatory process is modulated in part by oxidized fatty acids (oxylipins) that regulate all aspects of inflammation. Oxylipin profiles have been characterized in healthy periparturient cows, but their dynamics during CK are unknown. CK is an acute metabolic disease requiring clinical therapy, commonly including propylene glycol (PG) as a gluconeogenic agent. Recently, we showed that including lipolysis inhibitors such as niacin (NIA) and flunixin meglumine (FM) improved CK recovery. These drugs may modulate oxylipin biosynthesis by regulating the release of PUFA (oxylipin substrates) and cyclooxygenase activity. However, their impact on oxylipin profiles in cows with CK is unknown. The objective of this study was to determine the dynamics of specific linoleic and arachidonic acid-derived oxylipins during CK and following therapy with PG, NIA, and FM. Multiparous Jersey cows (n = 72; 7.1 DIM) with CK from a commercial dairy were sampled. Inclusion criteria were CK symptoms (lethargy, depressed appetite, and reduced rumen fill) and blood BHB ≥ 1.2 mmol/L. CK cows (n = 24/treatment) were randomly assigned to one of the 3 treatments: 1) PG:310 g orally once daily for 5 d, 2) PG+NIA:24 g orally once daily for 3 d, 3) PG+NIA+FM:1.1 mg/kg IV once daily for 3 d. Healthy cows (HC; n = 24) matched by lactation and DIM (±2 d) were also included. Plasma oxylipins were quantified at enrollment and 7d (d7) later using HPLC-MS/MS. At enrollment, CK had higher concentrations of arachidonic acid (AA) derived 5- and 20-HETE, 8,9-, 11,12-, and 14–15-DHET, and lower concentrations of linoleic acid (LA) derived 12,13-EpOME, 13-oxoODE 9,10- and 12,13-DiHOME. Integrated analysis of biological pathways and oxylipin profiles using Ingenuity Pathway Analysis (IPA) revealed AA metabolism as the top pathway activated during CK. By d7, treatment with PGNIAFM restored plasma PUFAs and oxylipins to profiles similar to HC. IPA analysis showed that PGNIAFM activated the zinc transporter SLC30A7, associated with reduced activation of the AA pathway. Results indicate that higher FA availability during CK, driven in part by dysregulated lipolysis, increases the pool of substrates for oxylipin biosynthesis. These oxylipins may play a role in both metabolic dysregulation and restoring homeostasis during CK. Inhibiting lipolysis and cyclooxygenase activity with NIA and FM can alter AA and LA-derived oxylipins biosynthesis. These findings underscore the potential use of lipolysis inhibitors NIA and FM in CK therapeutics and highlight the importance of understanding oxylipin pathways in the pathogenesis of CK.

Association of breast milk-derived arachidonic acid-induced infant gut dysbiosis with the onset of atopic dermatitis

ObjectiveThe specific breast milk-derived metabolites that mediate host–microbiota interactions and contribute to the onset of atopic dermatitis (AD) remain unknown and require further investigation.DesignWe enrolled 250 mother–infant pairs and collected...

RNA Sequencing Reveals the Inhibitory Effect of High Levels of Arachidonic Acid and Linoleic Acid on C2C12 Differentiation and Myogenic Biomarkers.

Over the past three decades, studies have shown that consuming polyunsaturated fatty acids (PUFAs) can enhance animal and human health and welfare through biological, biochemical, pathological, and pharmacological impacts. Furthermore, omega-6 plays key roles in the cardiopulmonary system, including promoting airway relaxation and inhibiting atherosclerosis and hypertension. However, findings from investigations of the effects of omega-6 fatty acids on molecular and cellular activity and discussions on their influence on biomarkers are still unclear. Therefore, the present study aimed to evaluate omega-6 fatty acids, the arachidonic acid (AA), and linoleic acid (LA) effects on C2C12 proliferation, myogenesis morphology, and relative myogenic biomarker expression through the Wnt pathway. C2C12 cells were cultured with and without 25, 50, 100, and 150 µM of LA and AA and then subjected to CCK8, Giemsa staining, RT qPCR, Western blotting, and RNA Sequencing. The CCK8 Assay results showed that 25, 50, 100, and 150 µM LA significantly decreased the viability after 72 h for 25, 50, 100, and 150 µM concentrations. Also, AA supplementation decreased cell viability after 24 h for 150 µM, 48 h for 150 µM, and 72 h for 50, 100, and 150 µM concentrations. Moreover, the LA and AA inhibitory effects noticed through Gimesa staining were morphological changes during myoblast differentiation. Both LA and AA showed inhibiting IGF1, Cola1, Col6a2, Col6a1, Itga10, Itga11, SFRP2, DAAM2, and NKD2 effects; however, the depressing effect was higher for AA compared to LA. The previous results were confirmed through Western blotting, which showed that 50 µM LA and AA significantly reduced DAAM2 and SFRP2 protein levels compared to the control. Regarding RNA sequencing results, LA and AA increased the number of differentially expressed (DE) Mt-rRNA and snoRNA; however, the numbers of lncRNA detected decreased compared to the control. Our findings demonstrate that high and moderate LA and AA concentrations reduce primary myoblast proliferation and differentiation. Also, they highlight novel biomarkers and regulatory factors to improve our understanding of how the nutrition of fatty acids can control and modulate the myogenesis and differentiation process through different biomarker families.

Flocculation of oleaginous green algae with Mortierella alpina fungi

Microalgae are promising sources of valuable bioproducts such as biofuels, food, and nutraceuticals. However, harvesting microalgae is challenging due to their small size and low biomass concentrations. To address this challenge, bio-flocculation of starchless mutants of Chlamydomonas reinhardtii (sta6/sta7) was investigated with Mortierella alpina, an oleaginous fungus with high concentrations of arachidonic acid (ARA). Triacylglycerides (TAG) reached 85 % of total lipids in sta6 and sta7 through a nitrogen regime. Scanning electron microscopy determined cell-wall attachment and extra polymeric substances (EPS) to be responsible for flocculation. An algal-fungal biomass ratio around 1:1 (three membranes) was optimal for bio-flocculation (80–85 % flocculation efficiency in 24 h). Nitrogen-deprived sta6/sta7 were flocculated with strains of M. alpina (NVP17b, NVP47, and NVP153) with aggregates exhibiting fatty acid profiles similar to C. reinhardtii, with ARA (3–10 % of total fatty acids). This study showcases M. alpina as a strong bio-flocculation candidate for microalgae and advances a mechanistic understanding of algal-fungal interaction.

The dose-response effects of arachidonic acid on primary human skeletal myoblasts and myotubes.

Cellular inflammatory response, mediated by arachidonic acid (AA) and cyclooxygenase, is a highly regulated process that leads to the repair of damaged tissue. Recent studies on murine C2C12 cells have demonstrated that AA supplementation leads to myotube hypertrophy. However, AA has not been tested on primary human muscle cells. Therefore, the purpose of this study was to determine whether AA supplementation has similar effects on human muscle cells. Proliferating and differentiating human myoblasts were exposed to AA in a dose-dependent manner (50-0.80µM) for 48 (myoblasts) or 72 (myotubes) hours. Cell viability was tested using a 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay and cell counting; myotube area was determined by immunocytochemistry and confocal microscopy; and anabolic signaling pathways were evaluated by western blot and RT-PCR. Our data show that the treatment of primary human myoblasts treated with 50µM and 25µM of AA led to the release of PGE2 and PGF2α at levels higher than those of control-treated cells (p <0.001 for all concentrations). Additionally, 50µM and 25µM of AA suppressed myoblast proliferation, myotube area, and myotube fusion. Anabolic signaling indicated reductions in total and phosphorylated TSC2, AKT, S6, and 4EBP1 in myoblasts at 50µM of AA (p <0.01 for all), but not in myotubes. These changes were not affected by COX-2 inhibition with celecoxib. Together, our data demonstrate that high concentrations of AA inhibit myoblast proliferation, myotube fusion, and myotube hypertrophy, thus revealing potential deleterious effects of AA on human skeletal muscle cell health and viability.

In pregnancy, maternal HDL is specifically enriched in, and carries the highest proportion of, DHA in plasma

Arachidonic acid (AA) and docosahexaenoic acid (DHA) are important for neurological development. The aim was to determine the distribution and relative enrichment of AA and DHA among lipoprotein fractions prior to pregnancy, throughout gestation and in the post-partum period. Our hypothesis was that in pregnancy, in contrast to the non-pregnant state, AA and DHA are carried in highest concentration in the very low density lipoprotein (VLDL) fraction secondary to increased gestational liver triglyceride secretion. Two independent prospective, observational cohort studies carried out in Glasgow were combined; one early in pregnancy and one later in pregnancy with post-partum follow up. Across the pregnancy timeline plasma lipoproteins were isolated using sequential ultracentrifugation and lipoprotein fatty acids were extracted and analysed by gas chromatography. High density lipoprotein (HDL) had the highest concentration of AA and DHA compared to other lipoproteins. HDL became progressively enriched in the proportion of triglycerides at 16 weeks of gestation, which peaked at 35 weeks and returned to baseline at 13 weeks postpartum. HDL DHA per HDL-cholesterol and HDL DHA per apoA-I became progressively enriched at 16 weeks of gestation, peaked at 25 weeks and returned to baseline at 13 weeks postpartum, whereas HDL AA (per HDL-C or HDL-apoA-I) did not differ. DHA is carried primarily in HDL rather than VLDL. HDL has anti-oxidant properties that might afford DHA protection against oxidation.

Arachidonic Acid Impairs Primary Human Skeletal Muscle Myoblasts Proliferation and Differentiation

Inflammation is a process initiated by cells in response to disease or injury. The cellular response to inflammation begins with the release of inflammatory cytokines such as TNF alpha and IL‐6. This cascade of inflammatory cytokines causes the release of arachidonic acid, a key regulator of inflammation, from the cell membrane, allowing an interaction with cyclooxygenase (COX) enzymes, mediating the synthesis of pro‐inflammatory prostaglandins (PGE2 and PGF2α). Recent studies have indicated that these inflammatory events, and particularly the release of arachidonic acid, have a major impact on how muscle cells and tissue respond to inflammation; these studies, conducted on murine C2C12 cells, demonstrated that arachidonic acid supplementation leads to increased muscle cell growth. We therefore sought to determine if similar arachidonic acid supplementation in both proliferating and differentiating primary human myoblasts leads to increased muscle cell growth. Proliferating and differentiating human myoblasts were exposed to arachidonic acid at concentrations from 50 μM‐ 0.80 μM for up to 48–72 hours respectively. Cell viability was tested using 3‐(4,5‐Dimethylthiazol‐2‐Yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) assay, myotube area was determined by immunocytochemistry and confocal microscopy, and anabolic signaling pathways were evaluated by Western blot and RTPCR. Our data suggests that treatment of primary human myoblasts with high concentrations of arachidonic acid (50 μM and 25 μM) leads to the release of PGE2 and PGF2α at levels higher than that of control treated cells (p&lt;0.001 for all concentrations). High concentrations of arachidonic acid have a deleterious effect on both myoblast proliferation, myotube area, and myotube fusion. Our data demonstrates that arachidonic acid reduced proliferation by 85% and 32%, myotube area by 63% and 38% and myotube fusion index by 93% and 50% respectively (P&lt;0.001 for all). As a potential mechanism for these decreases, we analyzed the AKT proliferation pathway and found a 90% and 37% reduction respectively in Eukaryotic translation initiation factor 4E‐binding protein 1(4EBP1) following the 48 hour arachidonic acid exposure at these concentrations (p&lt;0.001 and p&lt;0.05). Finally, as a potential mechanism for the change in myotube area and fusion index we analyzed the levels of myosin (specifically MYH 1,2,3,7, and 8), a protein critical for the formation of myoblasts into myotubes, by RTPCR. Our data suggests that 50 μM arachidonic acid reduced the expression of all analyzed MHY isoforms by an average of 95% (p&lt;0.001 for all) with normal expression levels not returning until the 3.125 μM dose. Together, our data demonstrates that high doses of arachidonic acid reduce primary human myoblast differentiation. These findings suggests that the response of primary human myoblasts are different than murine C2C12 cells supplemented with high doses of arachidonic acid.Support or Funding InformationThe views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. This abstract has been approved for public release with unlimited distribution.

Adipose tissue fatty acid and lipid mediator composition in obesity and response to chronic marine omega-3 fatty acid supplementation

AbstractIntroduction:Obesity is an excess of adipose tissue (AT) and is linked with increased inflammation that enhances risk of type-2 diabetes and cardiovascular disease. The BIOCLAIMS study assessed the fatty acid (FA) and lipid mediator composition in normal weight and obese individuals, and their response to chronic omega-3 FA supplementation.Materials and methods:AT biopsies were collected pre- and post-12 week supplementation with 1.1 g EPA + 0.8 g DHA/day or corn oil. The composition of FA in the total lipid extract (TLE) of AT from 37 normal weight and 44 obese subjects was assessed by gas chromatography (GC) and the concentration of lipid mediators in AT TLE of 36 normal weight and 45 obese subjects by coupled GC-mass spectrometry.Results:AT of obese subjects had higher concentrations of arachidonic acid (AA), EPA, DPA, PGF2α, arachidonoylethanolamine (AEA), and eicosapentaenoylethanolamine (EPEA) than that of normal weight subjects (P &lt; 0.05). Obese subjects also had and lower concentrations of mediators derived from linoleic and α-linolenic acids, DHA-derived mediators including RvD2 and hydroxydocosahexaenoic acids (HDHAs), AA-derived mediators including lipoxin-B4, hepoxilin-A3, 11,12-dihydroxyeicosatetrienoic acid (DHET), and 6-keto-PGF1α, and the FA ethanolamine of myristic acid and glycerol ester of palmitic acid (P &lt; 0.05 all) than normal weight subjects.Chronic supplementation with EPA + DHA increased the concentration of EPA (P = 0.006) and EPA-derived lipid mediators including dihydroxyeicosatetrienoic acids, EPEA, and EPA-glycerol ester (P &lt; 0.05), and increased the concentration of DHA (P &lt; 0.001), docosahexaenoylethanolamine, and 8-, 11-, 14-, and 16-HDHA (P &lt; 0.05). Chronic supplementation with EPA + DHA also decreased the concentration 4-, 17-, and 20-HDHA, and AA-derived lipid mediators including DHETs, AEA, and LTs in normal weight subjects (P &lt; 0.05), and decreased 2-AG in obese subjects (P &lt; 0.05).Discussion:These data indicate altered lipid signalling in AT in obesity (at baseline) suggesting dysregulation of adipose tissue expansion and inflammatory signalling, including lack of self-resolution, as well as dysregulation in the utilization of supplementary EPA + DHA for synthesis of anti-inflammatory lipid mediators. EPA + DHA are able to modulate synthesis of EPA-, DHA- and AA-derived lipid mediators but obesity may involve resistance to these effects particularly in endocannabinoid synthesis.

Arachidonic acid causes lysis of blood cells and ADP-dependent platelet activation responses in platelet function tests

The use of arachidonic acid (AA) to stimulate platelets is considered as a specific approach to study aspirin treatment efficacy. However, very high concentrations of AA are used, and it has been previously reported that AA can induce cell lysis in other settings. Several clinical studies have reported decreased responses to AA in whole blood tests in the presence of clopidogrel.Our aim was to investigate whether unspecific effects contribute to AA-induced aggregation and platelet activation in light transmission aggregometry (LTA) in platelet-rich plasma (PRP), and in assays using whole blood, multiple electrode aggregometry (MEA, Multiplate®), and flow cytometry.We report that cell lysis, especially of red blood cells, does occur at concentrations of AA used in the clinical tests and that ADP is very important for the AA-induced platelet activation responses. In flow cytometry, very limited platelet activation was detected before reaching AA concentrations in the millimolar range, where cell lysis also occurred, making it problematic to develop a reliable flow cytometry assay using AA as reagent.We conclude that cell lysis and ADP release contribute to AA-induced platelet responses, most markedly in whole blood assays. This finding could potentially explain some differences between studies comparing methods using whole blood and PRP and also how clopidogrel treatment could influence AA-induced aggregation results in previously published studies. Our findings highlight some issues with AA as reagent for platelet activation, which also have an impact on how platelet activation assays using AA should be interpreted.

Serum n-6 and n-9 Fatty Acids Correlate With Serum IGF-1 and Growth Up to 4 Months of Age in Healthy Infants.

The aim of this study was to study the relationship between insulin-like growth factor-1 (IGF-1), serum phospholipid fatty acids, and growth in healthy full-term newborns during infancy. Prospective observational study of a population-based Swedish cohort comprising 126 healthy, term infants investigating cord blood and serum at 2 days and 4 months of age for IGF-1 and phospholipid fatty acid profile and breast milk for fatty acids at 2 days and 4 months, compared with anthropometric measurements (standard deviation scores). At all time-points arachidonic acid (AA) was negatively associated with IGF-1. IGF-1 had positive associations with linoleic acid (LA) at 2 days and 4 months and mead acid (MA) showed positive associations in cord blood. Multiple regression analyses adjusted for maternal factors (body mass index, weight gain, smoking, education), sex, birth weight and feeding modality confirmed a negative association for the ratio AA/LA to IGF-1. MA in cord blood correlated to birth size. Changes in the ratios of n-6/n-3 and AA/docosahexaenoic acid from day 2 to 4 months together with infants' weight and feeding modality determined 55% of the variability of delta-IGF-1. Breast-fed infants at 4 months had lower IGF-1 correlating with lower LA and higher AA concentrations, which in girls correlated with lower weight gain from birth to 4 months of age. Our data showed interaction of n-6 fatty acids with IGF-1 during the first 4 months of life, and an association between MA and birth size when adjusted for confounding factors. Further follow-up may indicate whether these correlations are associated with later body composition.

Arachidonic acid causes hidden blood loss–like red blood cell damage through oxidative stress reactions

BackgroundHidden blood loss (HBL) often occurs in the prosthetic replacement for joint, but the mechanism is still not clear. Materials and methodsThis study tried to establish an animal model of HBL by injecting arachidonic acid (AA) into the Sprague-Dawley rats. Different concentrations of AA were injected into the tail veins of the rats, and blood samples were collected before and after administration at 24, 48, and 72 h. A complete blood count was obtained by to find the hemoglobin (Hb) and red blood cell (RBC) count changes. The glutathione peroxidase (GSH-PX) and total superoxide dismutase (T-SOD) activities and hydrogen peroxide (H2O2) levels were detected. The morphological changes of erythrocyte were observed under a polarizing microscope. The absorbance values of the blood samples were tested to determine the presence of ferryl Hb. ResultsHBL occurred in the experimental groups when the concentration of AA reached 10 mmol/L; Hb and RBC values decreased sharply at 24- and 48-h postinjection. This was followed by reduced activities of GSH-PX and T-SOD and decreased levels of H2O2. Moreover, the pathologic changes of red cell morphology mainly presented as pleomorphic RBC morphology, including cell rupture. The absorbance values of the blood samples were in accordance with ferryl Hb features. RBC and Hb values were relatively stable at 72 h. The GSH-PX and T-SOD activities and H2O2 levels gradually increased up to a balanced state. ConclusionsThe study concluded that high concentrations of AA can induce oxidative stress reactions in the body, causing acute injury of RBCs, which is closely related to HBL.

Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells.

It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.

Endocrine Disruptor DDE Associated with a High-Fat Diet Enhances the Impairment of Liver Fatty Acid Composition in Rats.

The banned pesticide dichlorodiphenyltrichloroethane (DDT) and its main metabolite, p,p'-dichlorodiphenyldichloroethylene (DDE), are commonly found in the food chain and in all tissues of living organisms. DDE is associated with metabolic diseases acting as an endocrine disruptor and more recently with the obesity pandemic. This study focuses on using fatty acid analysis to relate DDE exposure and metabolic dysfunction: liver and adipose tissue (visceral and subcutaneous) composition from male Wistar rats fed a standard (STD) or high-fat (HF) diet versus the addition of DDE in water. DDE exposure increased liver levels of palmitic, stearic, oleic, trans fatty, and linoleic acids having altered the n6 and n3 pathways leading to high concentrations of arachidonic acid and DHA (C22:6 n3). The results of this study confirm the close relationship between this pesticide metabolite and hepatic lipid dysfunction, underscoring its role as an emerging target for the prevention and therapy of nonalcoholic fatty liver disease (NAFLD).

Competition and allostery govern substrate selectivity of cyclooxygenase-2

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and its ester analog, 2-arachidonoylglycerol (2-AG), to prostaglandins (PGs) and prostaglandin glyceryl esters (PG-Gs), respectively. Although the efficiency of oxygenation of these substrates by COX-2 in vitro is similar, cellular biosynthesis of PGs far exceeds that of PG-Gs. Evidence that the COX enzymes are functional heterodimers suggests that competitive interaction of AA and 2-AG at the allosteric site of COX-2 might result in differential regulation of the oxygenation of the two substrates when both are present. Modulation of AA levels in RAW264.7 macrophages uncovered an inverse correlation between cellular AA levels and PG-G biosynthesis. In vitro kinetic analysis using purified protein demonstrated that the inhibition of 2-AG oxygenation by high concentrations of AA far exceeded the inhibition of AA oxygenation by high concentrations of 2-AG. An unbiased systems-based mechanistic model of the kinetic data revealed that binding of AA or 2-AG at the allosteric site of COX-2 results in a decreased catalytic efficiency of the enzyme toward 2-AG, whereas 2-AG binding at the allosteric site increases COX-2's efficiency toward AA. The results suggest that substrates interact with COX-2 via multiple potential complexes involving binding to both the catalytic and allosteric sites. Competition between AA and 2-AG for these sites, combined with differential allosteric modulation, gives rise to a complex interplay between the substrates, leading to preferential oxygenation of AA.

Plasma Phospholipid PUFAs Are Associated with Greater Muscle and Knee Extension Strength but Not with Changes in Muscle Parameters in Older Adults1–3

Muscle mass, intermuscular adipose tissue, and strength are important indicators of physical function. Dietary fatty acids (FAs) have been associated with muscle parameters such as larger size and higher strength, but large, population-based longitudinal data in older adults who are at risk of functional decline are lacking. The objective of this study was to investigate associations between plasma phospholipid polyunsaturated fatty acids (PUFAs) and measures of muscle size, intermuscular adipose tissue, and muscle strength cross-sectionally and after 5 y of follow-up. Data are from the Age, Gene/Environment Susceptibility-Reykjavik Study, a prospective cohort aged 66-96 y at baseline. The analytic sample included 836 participants with cross-sectional measures of muscle parameters and 459 participants with data on change in muscle parameters. PUFAs were assessed at study baseline through use of GC. Muscle parameters were assessed at baseline and after a median of 5.2 y. Muscle area and intermuscular adipose tissue were assessed with computed tomography. Maximal grip strength and knee extension strength were assessed with dynometers. Relative changes in muscle parameters (%) were calculated. Multivariate linear regression was performed to calculate unstandardized regression coefficients and P values for trends across tertiles of FAs are reported. Higher concentrations of total PUFAs were cross-sectionally associated with larger muscle size (P-trend: 0.002) and with greater knee extension strength (P-trend: 0.038). Higher concentrations of arachidonic acid were associated with smaller muscle size (P-trend: 0.015). Greater linoleic acid was associated with less intermuscular adipose tissue (P-trend: 0.004), whereas eicosapentaenoic acid (20:5n-3) was positively associated (P-trend: 0.047). Longitudinal analyses showed positive associations for α-linolenic acid with increased knee extension strength (P-trend: 0.014). No other associations were observed. These data illustrate the complex relation between plasma phospholipid PUFAs and muscle parameters; inconsistent cross-sectional relations with muscle size, intermuscular adipose tissue, and strength, and little evidence of a role in changes in muscle parameters.

Abstract 12357: Enzyme Kinetic Model Reveals Novel Characteristics of Pharmacodynamic Interaction of NSAIDs and Aspirin at COX-1, Which Lead to High on Aspirin Platelet Reactivity (HAPR)

Introduction: Non-aspirin NSAIDs attenuate the antiplatelet action of aspirin leading to High on Aspirin Platelet Reactivity (HAPR). However, detailed features of this interaction are still unresolved. Both competitive as well as non-competitive interaction at the cyclooxygenase-1 (COX-1) enzyme have been proposed. Hypothesis: We examined whether competitive enzyme kinetics describe COX-1 inhibition by aspirin and HAPR and if they are compatible with results obtained experimentally. Methods: We have developed an enzyme kinetic model of the aspirin/NSAID interaction at COX-1 using mathematical modelling and have varied enzyme kinetic parameters to predict the occurrence and the extent of HAPR. Subsequently, the predictions obtained were experimentally verified with selected NSAIDs using light transmission aggregometry and immunoassay for platelet thromboxane (TX) synthesis. Results: Enzyme kinetic modelling predicted that only low- and medium-affinity NSAIDs (Ki ≥1 μM), but not high-affinity NSAIDs, interfere with COX-1 inhibition by aspirin. Furthermore, the interaction was predicted to be most extensive at high concentrations of arachidonic acid (1mM). Experimental results confirmed the model by showing that low or medium-affinity COX-1 inhibitors piroxicam, dipyrone and celecoxib (each n=5-10) largely prevented the inhibition of aggregation and platelet TX synthesis in vitro by 30 μmol/L aspirin whereas the high-affinity COX-1 inhibitor SC560 (n=6) did not interfere with aspirin at all. In accordance to the mathematical model, aspirin/NSAID interaction was more pronounced in presence of high (1mmol/L) versus low (0.3mmol/L) concentration of arachidonic acid. Hence, the kinetic model was validated experimentally. Conclusion: Both enzyme kinetic modelling and experimental results suggest competitive antagonism of NSAID and aspirin at COX-1 as a cause for HAPR, possibly resulting in cardiovascular events. Critical parameters that influence this interaction are drug concentrations and NSAID affinity. Interestingly, the concentration of arachidonic acid also influences the NSAID/aspirin interaction, suggesting that “fatty acid tone” and lipid metabolism may influence this unfavourable drug/drug interaction.

The association between interpregnancy interval and birth weight: what is the role of maternal polyunsaturated fatty acid status?

BackgroundThe objective of this study was to evaluate the mediating role of maternal early pregnancy plasma levels of long chain polyunsaturated fatty acids (LCPUFAs) in the association of interpregnancy interval (IPI) with birth weight and smallness for gestational age (SGA) at birth.MethodsWe analysed a subsample of the Amsterdam Born Children and their Development (ABCD) cohort, comprising 1,659 parous pregnant women recruited between January 2003 and March 2004. We used linear and logistic regression to evaluate the associations between fatty acid status, interpregnancy interval and pregnancy outcome.ResultsLow plasma phospholipids concentrations of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and dihomo-gamma-linolenic acid (DGLA), and high concentrations of arachidonic acid (AA) during early pregnancy were associated with reduced birth weight and/or an increased risk of SGA. Short IPIs (< 6 months, with 18–23 months as a reference) were associated with a mean decrease of 207.6 g (SE: ± 73.1) in birth weight (p = 0.005) and a twofold increased risk of SGA (OR: 2.05; CI: 0.93–4.51; p = 0.074). Adjustment for maternal fatty acid concentrations did not affect these results to any meaningful extent.ConclusionsDespite the observed association of maternal early pregnancy LCPUFA status with birth weight and SGA, our study provides no evidence for the existence of an important role of maternal EPA, DHA, DGLA or AA in the association of short interpregnancy intervals with birth weight and SGA.

Arachidonic Acid Randomizes Endothelial Cell Motion and Regulates Adhesion and Migration

Cell adhesion and migration are essential for the evolution, organization, and repair of living organisms. An example of a combination of these processes is the formation of new blood vessels (angiogenesis), which is mediated by a directed migration and adhesion of endothelial cells (ECs). Angiogenesis is an essential part of wound healing and a prerequisite of cancerous tumor growth. We investigated the effect of the amphiphilic compound arachidonic acid (AA) on EC adhesion and migration by combining live cell imaging with biophysical analysis methods. AA significantly influenced both EC adhesion and migration, in either a stimulating or inhibiting fashion depending on AA concentration. The temporal evolution of cell adhesion area was well described by a two-phase model. In the first phase, the spreading dynamics were independent of AA concentration. In the latter phase, the spreading dynamics increased at low AA concentrations and decreased at high AA concentrations. AA also affected EC migration; though the instantaneous speed of individual cells remained independent of AA concentration, the individual cells lost their sense of direction upon addition of AA, thus giving rise to an overall decrease in the collective motion of a confluent EC monolayer into vacant space. Addition of AA also caused ECs to become more elongated, this possibly being related to incorporation of AA in the EC membrane thus mediating a change in the viscosity of the membrane. Hence, AA is a promising non-receptor specific regulator of wound healing and angiogenesis.

In-vitro platelet responses to arachidonic acid in the rat.

Using both the turbidimetric and the conductive methods to study aggregation of platelets, we found that arachidonic acid stimulated rat washed platelets in a dose-dependent manner (40 microM-0.5 mM). Although a high concentration of arachidonic acid (0.5 mM) produced an increase in light transmission both in the presence of 2 mM CaCl2 and EGTA (45.8 +/- 2.8 and 50.4 +/- 0.8% respectively) no changes in impedance were detected. Lysis caused by this concentration of arachidonic acid was very high at all the concentrations of calcium used (mean of 81.3%). In addition, the turbidimetric response induced by 0.5 mM arachidonic acid implied an initial decrease in light transmission but did not correlate with a real shape change. Forty micromolar arachidonic acid induced a calcium-dependent aggregation measured both by aggregometry and impedance. Morphology of aggregates induced by both concentrations was also studied. These results suggest that the optimal concentration for studying rat platelet activation by arachidonic acid is 40 microM; high concentrations (0.5 mM) cause aspecific effects not correlated to a physiological activation response.

Egg Fatty Acid Composition from Lake Trout Fed Two Lake Michigan Prey Fish Species

We previously demonstrated that there were significant differences in the egg thiamine content in lake trout Salvelinus namaycush fed two Lake Michigan prey fish (alewife Alosa pseudoharengus and bloater Coregonus hoyi). Lake trout fed alewives produced eggs low in thiamine, but it was unknown whether the consumption of alewives affected other nutritionally important components. In this study we investigated the fatty acid composition of lake trout eggs when females were fed diets that resulted in different egg thiamine concentrations. For 2 years, adult lake trout were fed diets consisting of four combinations of captured alewives and bloaters (100% alewives; 65% alewives, 35% bloaters; 35% alewives, 65% bloaters; and 100% bloaters). The alewife fatty acid profile had higher concentrations of arachidonic acid and total omega-6 fatty acids than the bloater profile. The concentrations of four fatty acids (cis-13, 16-docosadienoic, eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids) were higher in bloaters than in alewives. Although six fatty acid components were higher in lake trout eggs in 2001 than in 2000 and eight fatty acids were lower, diet had no effect on any fatty acid concentration measured in lake trout eggs in this study. Based on these results, it appears that egg fatty acid concentrations differ between years but that the egg fatty acid profile does not reflect the alewife-bloater mix in the diet of adults. The essential fatty acid content of lake trout eggs from females fed alewives and bloaters appears to be physiologically regulated and adequate to meet the requirements of developing embryos.