Doses Of Arachidonic Acid Research Articles

Effects of paternal arachidonic acid supplementation on offspring behavior and hypothalamus inflammation markers in the mouse.

Arachidonic acid (AA) is involved in inflammation and plays a role in growth and brain development in infants. We previously showed that exposure of mouse sires to AA for three consecutive generations induces a cumulative change in fatty acid (FA) involved in inflammation and an increase in body and liver weight in the offspring. Here, we tested the hypothesis that paternal AA exposure changes the progeny's behavioral response to a proinflammatory insult, and asked whether tissue-specific FA are associated with that response. Male BALB/c mice were supplemented daily with three doses of AA for 10 days and crossed to non-supplemented females (n = 3/dose). Two-month-old unsupplemented male and female offspring (n = 6/paternal AA dose) were exposed to Gram-negative bacteria-derived lipopolysaccharides (LPS) or saline control two hours prior to open field test (OFT) behavioral analysis and subsequent sacrifice. We probed for significant effects of paternal AA exposure on: OFT behaviors; individual FA content of blood, hypothalamus and hypothalamus-free brain; hypothalamic expression profile of genes related to inflammation (Tnfa, Il1b, Cox1, Cox2) and FA synthesis (Scd1, Elovl6). All parameters were affected by paternal AA supplementation in a sex-specific manner. Paternal AA primed the progeny for behavior associated with increased anxiety, with a marked sex dimorphism: high AA doses acted as surrogate of LPS in males, realigning a number of OFT behaviors that in females were differential between saline and LPS groups. Progeny hypothalamic Scd1, a FA metabolism enzyme with documented pro-inflammatory activity, showed a similar pattern of differential expression between saline and LPS groups at high paternal AA dose in females, that was blunted in males. Progeny FA generally were not affected by LPS, but displayed non-linear associations with paternal AA doses. In conclusion, we document that paternal exposure to AA exerts long-term behavioral and biochemical effects in the progeny in a sex-specific manner.

Arachidonic acid supplementation attenuates adipocyte inflammation but not adiposity in high fat diet induced obese mice

Obesity is associated with low-grade chronic inflammation and has a remarkable role in the pathophysiology of metabolic complications. In triggering these inflammatory responses, the arachidonic acid (AA) cascade plays a key role. However, there is a lack of data on how supplementary AA would affect obesity, adipose tissue inflammation, and the AA cascade in obesity. This study aims to investigate how AA supplementation affects obesity, adipocyte morphology, inflammation, and AA cascade signaling. Male Swiss Albino mice were used in our experiment. The mice were fed high-fat diets to induce obesity, and these obese mice were treated with two different doses of AA for 3 weeks. A normal diet non-obese group and an untreated obese group were kept as controls. Bodyweight and daily food intake data were recorded during that period. After the treatment period, blood serum and white adipose tissue of the experimental mice were collected for colorimetric lipid profile tests, histology, and mRNA extraction. The ΔΔCT method was employed for calculating the relative mRNA expression of target genes. The findings of our study suggest that AA has no significant effects on body weight, visceral adiposity, adipose tissue morphology, and serum lipid profile. However, AA treatment has resulted in a significant down-regulation of pro-inflammatory markers as well as the COX pathway. Besides, up-regulation of 12/15-LOX has been observed, indicating the metabolism pathway of supplementary AA through the LOX pathway. Our findings indicate that AA treatment may not provide significant benefits in terms of body weight, visceral fat mass, or serum lipid profile. However, it has effectively alleviated obesity-induced adipocyte inflammation in high-fat diet-induced obese mice.

Bone Marrow Adipocytes Induce Metabolic Reprogramming of Multiple Myeloma Cells

Obesity-induced increases in bone marrow adipocyte (BMAd) numbers and volume are associated with an increased risk of multiple myeloma (MM). We analyzed gene expression from previously published public datasets and found that 11 out of 47 genes associated with fatty acid (FA) metabolism showed increasing trend from MGUS (monoclonal gammopathy of undetermined significance), MM, to plasma cell leukemia. These genes included ACC1, the first and rate-limiting step of de novo fatty acid biosynthesis and ECHS1, the second step of the mitochondrial fatty acid beta-oxidation pathway. We have previously shown that adipocytes support the growth of MM cells. However, the molecular mechanisms of interaction between MM cells and BMAd remain largely unknown. Here, we hypothesize that BMAd support MM cells through metabolic reprogramming.Here we sought to identify the molecular pathways involved in MM/ BMAd interaction. BM aspirates of MGUS, smoldering MM (SMM), and newly diagnosed MM (NDMM) patients were used to isolate fat-enriched BM fraction and BM stromal cells (BMSCs). Murine BMSC cell-line OP9, murine MM cell-line 5TGM1, and human MM cell lines MM.1S and OPM2 were obtained from ATCC or provided by collaborators and cultured as their respective standard procedures. In-vitro adipogenesis was induced in OP9 cells or BMSCs by supplementing media with dexamethasone, indomethacin, insulin, and IBMX. MM cells were co-cultured directly with pre- or mature adipocytes. Cell proliferation was assessed using CyQUANT NF Cell Proliferation Assay. Lipolysis was assessed by High Sensitivity Lipolysis Assay Kit (Sigma). Lipid uptake in MM cells was assessed by flow-cytometry analysis of the incorporation of fluorescent 12- or 16-carbon long-chain fatty acids BODIPY-FL-C12 and BODIPY-FL-C16, respectively, or LipidTox labelled FA from co-cultured adipocytes. In-vivo effects of excess FA on MM cell growth were assessed using a plasmacytoma model in CB17 SCID mice.In-vitro co-culture revealed that BMSC-derived adipocytes (Ad) from MGUS, SMM and NDMM donors increased the proliferation of MM.1S MM cells significantly. Similarly, mature murine OP9 Ad cells also increased the proliferation of 5TGM1 murine MM cells. Interestingly, co-cultures showed dramatic decrease in LipidTox-stained lipid-droplet size distribution, suggesting increased lipolysis in Ad. In the process of lipolysis, various lipase enzymes hydrolyze stored triglycerides into free fatty acids (FFA) and glycerol. Co-culture of 5TGM1 or OPM2 cells with OP9 mature Ad increased glycerol secretion in the conditioned media by more than 3-fold indicating that MM induce lipolysis in Ad. Consequently, BMAd from MGUS, SMM, and NDMM patients showed increased expression of genes responsible for lipolysis (NR1H3) and increased FA desaturation (SCD1, FASD2). Although MM cells lacked intracellular lipid storage, OPM2 and 5TGM1 MM cells rapidly took up BODIPY-C12 and -C16 FAs. The FA secreted from Ad were directly taken up by MM cells as shown by transfer of LipidTox-labeled lipids from OP9 Ad to unstained 5TGM1 or OPM2 MM cells as assessed by flow cytometry. The addition of Acipomox, a small-molecule inhibitor of lipolysis, decreased LipidTox signal in MM cells compared to untreated OP9 cells. FA are primarily transported into cells through FATP (1-6) or CD36 receptors. Bioinformatic analysis of public database showed that FATP1 and FATP4 were highly expressed in 21 human MM cell lines. Indeed, MM cells from NDMM patients expressed high levels of FATP1 and FATP4. The uptake of BODIPY-C12 and -C16 by 5TGM1 or OPM2 MM cells was significantly reduced in the presence of Lipofermata, a pharmacological small-molecule inhibitor of FATP. Lipidomic analysis of BM aspirates from MM patients showed altered expression of various FA, including arachidonic acid (AA). Low doses of AA (0.125 - 2 µM) increased the proliferation and viability of MM cells whereas high doses (25- 100µM) dramatically decreased it, indicating a bimodal cellular effect of AA. Peritumoral AA treatment in a plasmacytoma model in CB17 SCID mice using MM.1S cells dramatically decreased tumor volume along with the markers of proliferation.In summary, we show that MM cells induce lipolysis in BMAd and that the released FFA are then taken up by MM cells through FATPs. Inhibition of either BMAd lipolysis or FFA transporter into MM cells could be a potential novel strategy to prevent MM progression. DisclosuresFulzele: Constellation Pharma: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company. Raje: Celgene, Amgen, Bluebird Bio, Janssen, Caribou, and BMS: Other.

Arachidonic acid drives adaptive responses to chemotherapy-induced stress in malignant mesothelioma

BackgroundHigh resistance to therapy and poor prognosis characterizes malignant pleural mesothelioma (MPM). In fact, the current lines of treatment, based on platinum and pemetrexed, have limited impact on the survival of MPM patients. Adaptive response to therapy-induced stress involves complex rearrangements of the MPM secretome, mediated by the acquisition of a senescence-associated-secretory-phenotype (SASP). This fuels the emergence of chemoresistant cell subpopulations, with specific gene expression traits and protumorigenic features. The SASP-driven rearrangement of MPM secretome takes days to weeks to occur. Thus, we have searched for early mediators of such adaptive process and focused on metabolites differentially released in mesothelioma vs mesothelial cell culture media, after treatment with pemetrexed.MethodsMass spectrometry-based (LC/MS and GC/MS) identification of extracellular metabolites and unbiased statistical analysis were performed on the spent media of mesothelial and mesothelioma cell lines, at steady state and after a pulse with pharmacologically relevant doses of the drug. ELISA based evaluation of arachidonic acid (AA) levels and enzyme inhibition assays were used to explore the role of cPLA2 in AA release and that of LOX/COX-mediated processing of AA. QRT-PCR, flow cytometry analysis of ALDH expressing cells and 3D spheroid growth assays were employed to assess the role of AA at mediating chemoresistance features of MPM. ELISA based detection of p65 and IkBalpha were used to interrogate the NFkB pathway activation in AA-treated cells.ResultsWe first validated what is known or expected from the mechanism of action of the antifolate. Further, we found increased levels of PUFAs and, more specifically, arachidonic acid (AA), in the transformed cell lines treated with pemetrexed. We showed that pharmacologically relevant doses of AA tightly recapitulated the rearrangement of cell subpopulations and the gene expression changes happening in pemetrexed -treated cultures and related to chemoresistance. Further, we showed that release of AA following pemetrexed treatment was due to cPLA2 and that AA signaling impinged on NFkB activation and largely affected anchorage-independent, 3D growth and the resistance of the MPM 3D cultures to the drug.ConclusionsAA is an early mediator of the adaptive response to pem in chemoresistant MPM and, possibly, other malignancies.

OAB-029: Bone marrow adipocytes induce metabolic reprogramming of multiple myeloma cells

<h3>Background</h3> Obesity-induced increases in bone marrow adipocyte (BMAd) numbers and volume are associated with an increased risk of MM. However, the associated molecular mechanisms have remained largely unknown. We hypothesize that BMAd support MM cells through metabolic reprogramming. <h3>Methods</h3> BM aspirates of MGUS, SMM, and NDMM patients were used to obtain mature BMAd or stromal cells (BMSCs). Murine BMSC cell-line OP9, MM cell-line 5TGM1, and human-origin MM cell-lines MM.1S, OPM2 were obtained from ATCC. Cell proliferation was assessed by CyQUANT Assay; lipolysis by Lipolysis Assay (Sigma); lipid uptake by flow cytometry (FCM) of fluorescent fatty acids (FA) BODIPY-C12 and -C16, or LipidTox. In-vivo efficacy of FA treatment was assessed in a MM CB17 SCID mouse plasmacytoma model. <h3>Results</h3> In-vitro co-culture revealed that BMSC-derived adipocytes (Ad) from MGUS/SMM and NDMM donors significantly increased the proliferation of MM.1S MM cells. Similarly, mature murine OP9 Ad cells also increased the proliferation of 5TGM1 murine MM cells. Interestingly, co-cultures showed dramatic decrease in LipidTox-stained lipid-droplet size distribution, suggesting increased lipolysis in Ad, further confirmed by increased glycerol content in conditioned media in the presence of MM cells. Moreover, BMAd from MGUS, SMM, and NDMM patients showed increased expression of genes responsible for lipolysis (NR1H3) and increased FA desaturation (SCD1, FASD2). Although MM cells lacked intracellular lipid storage, OPM2 and 5TGM1 MM cells rapidly took up BODIPY-C12 and -C16 FAs. The FA secreted from Ad were directly taken up by MM cells as shown by transfer of LipidTox-labeled lipids from OP9 Ad to unstained 5TGM1 or OPM2 MM cells as assessed by FCM. The addition of acipomox, a small-molecule inhibitor of lipolysis, decreased LipidTox signal in MM cells compared to untreated OP9 cells. FA are transported into cells through FATP (1-6) or CD36 receptors. Bioinformatics analysis of public Oncomine database showed that FATP1 and FATP4 were highly expressed in 21 human MM cell lines. MM cells from NDMM patients expressed high levels of FATP1 and FATP4. The uptake of BODIPY-C12 and -C16 by 5TGM1 or OPM2 MM cells was significantly reduced in the presence of Lipofermata, a pharmacological small-molecule inhibitor of FATP. Lipidomic analysis of BM aspirates from MM patients showed altered expression of various FA, including arachidonic acid (AA). Low doses of AA (0.125 - 2 µM) increased the proliferation and viability of MM cells whereas high doses (25- 100µM) dramatically decreased it, indicating a bimodal cellular effect of AA. Peritumoral AA treatment in a plasmacytoma model decreased tumor volume significantly. <h3>Conclusion</h3> We show that MM cells induce lipolysis in BMAd and that the released FFA are then taken up by MM cells through FATPs. Inhibition of either BMAd lipolysis or FFA transporter into MM cells could be a potential novel strategy to prevent MM progression.

Performance Metrics of the AGGRESTAR PL-12® Platelet Function Analyser in Patients with TIA or Ischaemic Stroke on Commonly-Prescribed Antiplatelet Regimens

Introduction: The optimal time interval after venepuncture to perform platelet function/reactivity testing at low shear stress on the novel AGGRESTAR PL-12® platelet function analyser in non-Chinese Cerebrovascular Disease (CVD) patients is unknown. Methods: Twelve TIA/ischaemic stroke patients were recruited to this cross-sectional, methodological study: 3 on aspirin monotherapy, aspirindipyridamole combination therapy, clopidogrel monotherapy and aspirinclopidogrel combination therapy, respectively. The PL-12 (‘mode 2’) was used to calculate the % maximum aggregation rate to fixed doses of arachidonic acid (%MARAA) and adenosine diphosphate (%MARADP). Samples were analysed every 15 minutes from 30-135 minutes, and every 30 minutes between 165-225 minutes after venepuncture to calculate the time interval providing optimal interassay Coefficients of Variation (CVs). Results: Mean CVs were ≤ 7.37% for the %MARAA assay in patients on aspirin monotherapy or combination therapy, and ≤ 10.24% for the %MARADP assay in patients on clopidogrel monotherapy or combination therapy if assays were performed between 90-120 minutes post-venepuncture. CVs ≤ 10% were also obtained from assays performed between 90-165 minutes postvenepuncture on aspirin monotherapy or combination therapy. Discussion: Reliable and reproducible platelet function/reactivity data can be obtained with the AGGRESTAR PL-12 analyser in non-Chinese CVD patients on commonly-prescribed antiplatelet monotherapy or combination therapy regimens between 90-120 minutes post-venepuncture.

PUFAs, BDNF and lipoxin A4 inhibit chemical-induced cytotoxicity of RIN5F cells in vitro and streptozotocin-induced type 2 diabetes mellitus in vivo

ObjectiveTo study whether minimal doses of arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and lipoxin A4 (LXA4) and brain-derived neurotrophic factor (BDNF), when used in combination can protect RIN5F cells from chemical-induced cytotoxicity. As a corollary, to know whether plasma BDNF and LXA4 are altered in STZ-induced type 2 DM animals.Materials and methodsRIN5F cells, alloxan (AL), streptozotocin (STZ), doxorubicin (DB), and benzo(a)pyrene (BP) were used in this study. Chemical-induced apoptosis and changes in antioxidants, lipid peroxides and nitric oxide (NO) and LXA4 and BDNF levels in RIN5F cells were studied. Alterations in plasma concentrations of BDNF and LXA4 in STZ-induced type 2 diabetes animals was estimated.ResultsBDNF, LXA4 and AA, EPA and DHA protected (P < 0.001 and P < 0.01 respectively) against AL/STZ/DB/BP-induced toxicity to RIN5F cells in vitro. AL/ STZ/DB/BP inhibited BDNF and LXA4 production by RIN5F cells and were restored to normal by AA, EPA and DHA. Sub-optimal doses of BDNF, LXA4, AA and EPA when used in combination protected against cytotoxic action of AL/STZ/DB/BP on RIN5F cells in vitro by restoring LXA4/BDNF levels and altered antioxidant/lipid peroxides/NO levels (P < 0.01) to normal. STZ (65 mg/kg)-induced type 2 diabetes mellitus animals showed reduced plasma BDNF and LXA4 levels (P < 0.001).DiscussionAL/STZ/DB/BP-induced cytotoxicity to RIN5F cells in vitro can be prevented by BDNF, LXA4 and AA. AL/STZ/DB/BP are cytotoxic, possibly, by suppressing the production of LXA4 and BDNF in RIN5F cells. STZ-induced type 2 DM animals have decreased plasma levels of LXA4 and BDNF.ConclusionThe results of the present study suggest that BDNF, LXA4, EPA, DHA, AA, GLA and BDNF protect pancreatic β cells from the cytotoxic action of various chemicals and prevent development of diabetes mellitus. LXA4 seems to be the mediator of these cytoprotective actions of BDNF and PUFAs suggesting a close interaction exists among these molecules (BDNF, PUFAs and LXA4). Hence, methods developed to deliver a combination of PUFAs (especially AA), LXA4 and BDNF may prevent development of diabetes mellitus (both type 1 and type 2).

Polyunsaturated Fatty Acid (PUFA) Signaling Induces Ferroptosis-Mediated Cell-Death in Multiple Myeloma

Altered cellular metabolic pathways are the hallmark of tumor cells. Multiple myeloma (MM) is positively correlated with metabolic disorders such as obesity and Gaucher's disease. The local bone marrow (BM) microenvironment (TME) majorly influences the initiation and progression of MM. In a typical MM patient, BM adipocytes make up 70% of the cellular volume. The abundance of adipocyte-secreted free fatty acids (FFA) may shift myeloma cellular metabolism from aerobic glycolysis to more energy-producing fatty acid oxidation. The FFAs are important catalysts of key downstream drug-targetable signaling pathways such as cyclooxygenase (COX), cytochrome P450 (CYP), and lipoxygenase (LOX) pathways. In this study, we hypothesized that altered lipid profile in the local BM TME contributes to MM progression. BM-Fat enriched tissue isolated from BM aspirates of Monoclonal Gammopathy of Undetermined Significance (MGUS) and smoldering MM (SMM) patients showed a significant increase in adipogenic PPARγ gene expression compared to aged-matched healthy donors (N≥3). The BM mesenchymal stem/progenitor cells (BMSCs) from MGUS/SMM patients expressed normal levels of BMSC markers CD271, CD105, CD44, CD106, CD29, CD90, CD49e, and Notch4 but showed significantly increased expression of adipogenic markers including Preadipocyte factor 1, Leptin Receptor, and Perilipin A (N=6). This also translated into significantly increased adipogenic differentiation of patient BMSCs when cultured alone or with the human MM cell-line MM.1S (N≥3). Furthermore, MM.1S showed significantly increased proliferation when co-cultured with BMSCs from MGUS/SMM patients (N=5). These data demonstrate a vicious cycle where adipogenesis is increased in early precursor MM stages that further support the growth of myeloma cells. We performed gas chromatometry based lipidomics analysis on the supernatant of BM aspirates from MGUS, SMM, and newly diagnosed MM (NDMM) patients. The analysis identified significant decreases in key polyunsaturated fatty acids (PUFA) including Arachidonic Acid (AA) and Docosatetraenoic acid (N≥5). Lipid metabolism specific gene array on RNA from adipose tissue fraction of BM aspirates from MGUS, SMM and NDMM patients showed altered changes in genes responsible for fatty acid synthesis and metabolism. PUFA are involved in anti-inflammatory mechanisms in cancer. We hypothesized that increased levels of certain PUFA, such as AA, in the BM TME may decrease MM progression. To test this hypothesis, we treated MM cells with physiological doses of AA. AA dose-dependently decreased proliferation and viability of human MM cell lines, MM1S, H929, and U266, and CD138+ patient myeloma cells. For in vivo studies, humanized MM tumor model was generated in SCID mice by growing MM.1S cells in the intrascapular subcutaneous region for 3-weeks. Mice were then treated with daily localized injections of vehicle, 100µg/g AA, 500µg/g of AA, or IV with 2mg/kg/biweekly Carfilzomib (CFZ), or CFZ with 500µg/g of AA (COMBO). Tumor volume significantly decreased in 500µg/g AA treatment group beginning 10-days and was comparable to the CFZ treatment. Gross examination and flow cytometry analysis of CD138+ myeloma cells showed dramatically increased tumor-cell apoptosis in 500µg/g AA and COMBO treatment groups. To identify the primary apoptosis-inducing AA signaling pathway in MM cells, we used specific inhibitors of each of these signaling pathways including ibuprofen (Cox inhibitor), baicalein (12-LOX inhibitor), BW B70C (5,15-LOX inhibitor), 1-aminobenzotriazole (CYP450 inhibitor), and ferrostatin (Ferroptosis/lipid peroxidation inhibitor). Among these compounds, ferrostatin treatment completely rescued AA induced apoptosis in the human MM.1S cells. Ferroptotic cell death is the result of an accumulation of lipid peroxides which is generally prevented by the enzyme Glutathione peroxidase 4 (GPX4). We, therefore investigated the role of AA on GPX4 and found that all MM cell lines partially or completely lost the expression of GPX4 when exposed to AA and that this effect was completely prevented when cotreated with Ferrostatin. Taken together, we show that BM adipocytes promote myeloma cell proliferation at least in part through secreted FFAs. Therapeutically targeting members of this signaling pathway, such as ferroptosis, is a potential novel treatment strategy for MM especially in the MGUS and SMM stages. Disclosures Raje: Celgene Corporation: Consultancy; Amgen Inc.: Consultancy; Bristol-Myers Squibb: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Merck: Consultancy.

A systematic review of the effects of increasing arachidonic acid intake on PUFA status, metabolism and health-related outcomes in humans.

We conducted a systematic review of randomised controlled trials (RCT) of increased intake of arachidonic acid (ARA) on fatty acid status and health outcomes in humans. We identified twenty-two articles from fourteen RCT. Most studies were conducted in adults. These used between 80 and 2000 mg ARA per d and were of 1-12 weeks duration. Supplementation with ARA doses as low as 80 mg/d increased the content of ARA in different blood fractions. Overall there seem to be few marked benefits for adults of increasing ARA intake from the typical usual intake of 100-200 mg/d to as much as 1000 mg/d; the few studies using higher doses (1500 or 2000 mg/d) also report little benefit. However, there may be an impact of ARA on cognitive and muscle function which could be particularly relevant in the ageing population. The studies reviewed here suggest no adverse effects in adults of increased ARA intake up to at least 1000-1500 mg/d on blood lipids, platelet aggregation and blood clotting, immune function, inflammation or urinary excretion of ARA metabolites. However, in many areas there are insufficient studies to make firm conclusions, and higher intakes of ARA are deserving of further study. Based on the RCT reviewed, there are not enough data to make any recommendations for specific health effects of ARA intake.

Essentiality of arachidonic acid intake in murine early development

We previously reported the importance of long-chain polyunsaturated fatty acid (LC-PUFA (>C20)) intake, including arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), for growth. This follow-up study focuses on ARA using a novel artificial rearing model during the lactation period in delta-6-desaturase knockout (D6D-KO) mice. Newborn D6D-KO male mouse pups were separated from dams within 48 hours and fed artificial milks containing 18-C essential fatty acids (EFAs) (16–17% LA, 3.8–4.1% ALA) with or without 1.2% ARA. After weaning, mice were maintained on similar diets: 15% LA, 2.3–2.4% ALA with or without 1.9% ARA. As a reference group, new born wild type (WT) male mouse pups were maintained by artificial milk and diet containing LA and ALA without ARA. Aspects of brain function were measured behaviorally (motor activity and rota-rod test) when mice were age 9 weeks. Body weight in the KO-Cont group was significantly lower (approximately 30%) than in the WT-Cont group, but this decrease was ameliorated by providing ARA in the KO-ARA group. The motor activity and coordination in the KO-Cont group decreased markedly compared to the WT-Cont group. The KO-ARA group had a tendency toward deteriorated motor coordination, although the motor activity was significantly enhanced compared to the KO-Cont group. In KO-ARA group brains, the level of ARA was increased and DHA decreased compared to WT-Cont. These results suggest that intake of LA and ALA only is insufficient to support healthy growth, and that ARA is also required, at least during the lactation period. These findings also suggested that continued intake of relatively high levels of ARA and without supplemental DHA during development led to an increased motor activity above that of WT animals. These studies indicate that both ARA dose and proper combination with DHA must be delineated to define optimal growth and behavioral function.

Impaired Social Interaction in Autism Spectrum Disorders

Autism spectrum disorders (ASD) are characterized by impairment in social reciprocity, disturbances in language and communication, restricted interests and repetitive behaviors of various types, as defined by the DSM-IV [1]. Currently, no drug has been consistently proven to be effective for the core social and communication impairment so central to the PDDs. Pharmacotherapy in ASD lacks a solid, reliable neurobiological basis and at present it is mainly directed at the socalled associated behavioral symptoms, with limited relevance to core symptoms. Atypical neuroleptics, especially risperidone, have been shown to be useful in the treatment of behavioral symptoms in ASD. Recent trials with SSRIs did not show remarkable results, in spite of their promising potential role [2,3]. Research is now directed at evaluating novel treatments and combined behavioral and pharmacologic treatments, since behavioral interventions are the mainstay of the early treatment of autism. Abnormal functional connectivity, which affects the delivery of afferent signals, may be involved in the pathophysiology of ASD [4]. Alternatively, individuals with ASD may be more vulnerable to oxidative stress due to deficit antioxidant defense mechanisms [5]. Polyunsaturated fatty acids, arachidonic acid (ARA) and Docosahexaenoic acid (DHA) play key roles in brain network maturation. ARA is important for signal transduction [6]. Therefore, it is clear that supplements containing antioxidants that are related to lipid peroxidation may be beneficial for individuals with ASD [7]. Previous double-blind randomized placebo-controlled studies have helped to validate the efficacy of DHA or eicosapentaenoic Acid (EPA) plus small doses of ARA (42 or 40 mg/day) in reducing behavioral problems in Attention-Deficit/Hyperactive Disorders (AD/HD) [8] and learning difficulties (LD) [9]. However, there are few studies that have examined the effects of larger doses of ARA added to DHA on the core behavioral and social impairments of ASD with double-blind, randomized, placebo-controlled designs. We therefore examined the effect of supplementation with large doses of ARA added to DHA (240 mg/day) in individuals with ASD (n=13) in a double-blind, placebocontrolled trial, followed by an open-label treatment. Daily doses of ARA and DHA were 240 mg/day. Four participants aged 6-10 years received one-half of the daily doses of the supplementation (for a total of 120 mg ARA and 120 mg DHA per day). Previous studies of supplementation with PUFAs, ARA doses were 40 mg/day [9] or 42 mg/day [8]. ARA doses used in this study were 120 or 240 mg/day, and our supplementation included thus larger doses of ARA compared to these previous studies.

Effects of Adding Large Doses of Arachidonic Acid to Docosahexaenoic Acid on Social Impairment in Individuals with Autism Spectrum Disorders

Autism spectrum disorders (ASD) are a cluster of developmental disorders, whose core symptoms can be attributed to abnormal development of the large-scale neural networks required for social communication. The impaired delivery of afferent signals has been proposed to be associated with impaired social interaction in ASD. Arachidonic acid (ARA) and docosahexaenoic acid (DHA) play key roles in the maturation of brain network. In particular, ARA promotes signal transduction related to neural function. It is therefore possible that supplementation with larger ARA doses added to DHA may mitigate the impaired social interaction observed in ASD. There have been no double-blind randomized placebo-controlled studies of the effect of large doses of ARA, added to DHA, on the impaired social interactions observed in ASD. This article presents the results of a 16-week double-blind randomized placebo-controlled trial in which high-dose ARA was added to DHA supplementation in individuals with ASD (n=13). The primary outcomes were measured using the Social Responsiveness Scale (SRS) and the secondary outcome measure was Autism Diagnostic Interview-revised (ADI-R) social and communication domains. Supplementation of larger doses of ARA added to DHA significantly improved SRS-measured communication and ADI-measured social communication. At the end of trial, the increase in plasma ARA levels from the baseline observed in the ARA treatment group was significantly higher compared to placebo group. Keywords: Autism spectrum disorders, impaired social interaction, arachidonic acid, dietary supplementation, clinical trial, signal transduction

Effects of Adding Large Doses of Arachidonic Acid to Docosahexaenoic Acid on Social Impairment in Individuals with Autism Spectrum Disorders

Autism spectrum disorders (ASD) are a cluster of developmental disorders, whose core symptoms can be attributed to abnormal development of the large-scale neural networks required for social communication. The impaired delivery of afferent signals has been proposed to be associated with impaired social interaction in ASD. Arachidonic acid (ARA) and docosahexaenoic acid (DHA) play key roles in the maturation of brain network. In particular, ARA promotes signal transduction related to neural function. It is therefore possible that supplementation with larger ARA doses added to DHA may mitigate the impaired social interaction observed in ASD. There have been no double-blind randomized placebo-controlled studies of the effect of large doses of ARA, added to DHA, on the impaired social interactions observed in ASD. This article presents the results of a 16-week double-blind randomized placebo-controlled trial in which high-dose ARA was added to DHA supplementation in individuals with ASD (n=13). The primary outcomes were measured using the Social Responsiveness Scale (SRS) and the secondary outcome measure was Autism Diagnostic Interview-revised (ADI-R) social and communication domains. Supplementation of larger doses of ARA added to DHA significantly improved SRS-measured communication and ADI-measured social communication. At the end of trial, the increase in plasma ARA levels from the baseline observed in the ARA treatment group was significantly higher compared to placebo group. Keywords: Autism spectrum disorders, impaired social interaction, arachidonic acid, dietary supplementation, clinical trial, signal transduction

Arachidonic acid supplementation during gestational, lactational and post-weaning periods prevents retinal degeneration induced in a rodent model

Fatty acids and their derivatives play a role in the response to retinal injury. The effects of dietary arachidonic acid (AA) supplementation on N-methyl-N-nitrosourea (MNU)-induced retinal degeneration was investigated in young Lewis rats during the gestational, lactational and post-weaning periods. Dams were fed 0·1, 0·5 or 2·0% AA diets or a basal (< 0·01% AA) diet. On postnatal day 21 (at weaning), male pups received a single intraperitoneal injection of 50 mg MNU/kg or vehicle, and were fed the same diet as their mother for 7 d. Retinal apoptosis was analysed by the terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick-end labelling (TUNEL) assay 24 h after the MNU treatment, and retinal morphology was examined 7 d post-MNU. Histologically, all rats that received MNU and were fed the basal and 0·1% AA diets developed retinal degeneration characterised by the loss of photoreceptor cells (disappearance of the outer nuclear layer and the photoreceptor layer) in the central retina. The 0·5 and 2·0% AA diets rescued rats from retinal damage. Morphometrically, in parallel with the AA dose (0·5 and 2·0% AA), the photoreceptor ratio significantly increased and the retinal damage ratio decreased in the central retina, compared with the corresponding ratios in basal diet-fed rats. In parallel with the increase in serum and retinal AA levels and the AA:DHA ratio, the apoptotic index in the central retina was dose-dependently decreased in rats fed the 0·5 and 2·0% AA diets. In conclusion, an AA-rich diet during the gestation, lactation and post-weaning periods rescued young Lewis rats from MNU-induced retinal degeneration via the inhibition of photoreceptor apoptosis. Therefore, an AA-enriched diet in the prenatal and postnatal periods may be an important strategy to suppress the degree of photoreceptor injury in humans.

Efficacy and mechanism of action of arachidonic acid in the treatment of hamsters infected with Schistosoma mansoni or Schistosoma haematobium

We have recently shown that in vitro and in vivo exposure of Schistosoma mansoni and Schistosoma haematobium to 5–10mM arachidonic acid (ARA) induces parasite surface membrane disintegration and eventual attrition. Here we report on the optimum ARA dose and post-infection treatment time for maximum schistosome demise in hamsters. A series of four experiments for each schistosome species indicated that oral administration of ARA after patency led to a highly significant (P<0.02 to <0.001) reduction in worm burden accompanied by a significant (P<0.05) decrease in worm egg load. ARA-mediated attrition in vivo appeared to be associated with high titres of serum antibodies to tegumental antigens. In support, serum antibodies from patently infected and ARA-treated hamsters readily bound to the surface membrane of ARA-exposed adult worms, as judged by indirect membrane immunofluorescence. More importantly, addition of serum antibodies and peripheral blood mononuclear cells significantly enhanced ARA-mediated adult worm attrition in vitro. These data together show that the schistosomicidal effect of ARA in laboratory animals is enhanced by immune effectors and is highly efficacious and entirely safe.

Efficacy of Adding Large Doses of Arachidonic Acid to Docosahexaenoic Acid against Restricted Repetitive Behaviors in Individuals with Autism Spectrum Disorders: A Placebo-Controlled Trial

Objectives: It has been documented that behavioral addictions resemble substance addiction in many domains, including the repetitive patterns of interests and behavior displayed by addicts and their failure to resist impulses to perform an act that are harmful to themselves or others. Restricted and repetitive patterns of interests and behavior resembling the characteristics of behavioral addictions are also a core feature of autism spectrum disorders (ASD). Impaired delivery of afferent signals has been proposed to be a factor in pathophysiology of ASD. The polyunsaturated fatty acids, arachidonicacid (ARA) and docosahexaenoic acid (DHA) play key roles in the maturation of the brain network. Supplementation of larger doses of ARA added to DHA may therefore improve repetitive and addictive behavior. Methods: To estimate the effi cacy of this supplementation in individuals with ASD, we conducted a 16-week double-blind, randomized placebo-controlled trial in 13 individuals with ASD. The outcomes were measured using the Aberrant Behavior Checklist (ABC), and the repetitive behavior subdomains of the Autism Diagnostic Interview-Revised (ADI-R). To study the mechanisms underlying the effects of this supplementation regimen, we examined the plasma levels of main polyunsaturated fatty acids (i.e., eicosapentaenoic acid, DHA and ARA). Results: Our supplementation regimen signifi cantly improved ABC-measured social withdrawal and ADI-R C3 subdomain (stereotyped and repetitive motor mannerisms). Their scores of the ADI-R C3 subdomain were signifi cantly correlated with ABC-measured inappropriate speech (r=0.397, P<0.01).The changes in the plasma ARA levels at the end of the placebo-controlled trial was signifi cantly increased in the treatment group. Conclusion: The observed improvement in repetitive and addictive behavior might have been related to reduction in the scores of the ABC-measured social withdrawal and ABC-measured inappropriate speech. These fi ndings suggest that improvements in impaired social interactions may contribute to improvements in repetitive and addictive behavior.

In vitro effects of arachidonic acid on immune functions of head kidney macrophages isolated from large yellow croaker (Larmichthys crocea)

The experiments were conducted to determine the effects of arachidonic acid (ARA) on macrophages isolated from large yellow croaker head kidney. After exposing to serum-free medium for 1day, cultured cells were incubated in medium supplemented with graded levels of ARA (0, 5, 25, 100, 200 and 1000μM) in the form of fatty acid bovine serum albumin (FA-BSA) complex for 12h, 24h and 36h, respectively. Cells treated with 0μM ARA (2% bovine serum albumin, BSA) were included as control at each incubation time. Following stimulation, cell viability, lipid peroxidation, secretory phopholipase A2 (sPLA2) production and some immune parameters including phagocytosis, respiratory burst activity, prostaglandin E2 (PGE2) and Interleukin 1β (IL-1β) production were examined. Results showed that cell viability decreased with prolonged exposure and increasing ARA concentration. Especially after 36h, cell viability was significantly decreased by incubation with higher levels of ARA (200 and 1000μM) (P<0.01). In the meanwhile, higher levels of ARA (200 and 1000μM) caused a significant increase in the production of malondialdehyde (MDA) (P<0.05, P<0.01). Phagocytosis percentage (PP) values were significantly higher in treatments with 25 and 100μM ARA (P<0.05), but significantly lower in treatments with 1000μM ARA compared to the control group (P<0.01). Enhanced intracellular superoxide anion (O2−) synthesis was found in cells treated with each level of ARA with an exception of 1000μM ARA (P>0.05). The significantly promoted production of sPLA2 seemed to be irrelevant to increased ARA concentration, while PGE2 production was significantly increased in an ARA dose dependent way (P<0.01). No pronounced effect on IL-1β production was observed between the control and ARA group, and levels of IL-1β in cell culture supernatant were fairly low (only approximately 6pg/ml). These findings suggested that ARA could influence the immunity and physiological conditions of macrophages from head kidney of large yellow croaker in vitro.

Central mechanism underlying pressor and bradycardic effect of intracerebroventricularly injected arachidonic acid

The aim of the current study was to determine the central cyclooxygenase (COX) pathway and central thromboxane signaling in the cardiovascular effects evoked by arachidonic acid (AA). As a main control for the study, different doses of AA (75, 150, or 300µg) were administered intracerebroventricularly (i.c.v.). Centrally injected AA dose- and time-dependently increased mean arterial pressure and decreased heart rate in conscious normotensive Sprague-Dawley rats. The maximal cardiovascular effects of AA were observed at min 10 of the injection and lasted almost 30min. To investigate the central mechanism of the AA-induced cardiovascular effect in conscious normotensive animals, pretreatment with nonselective COX inhibitor indomethacin (200µg; i.c.v.), thromboxane A2 (TXA2) synthesis inhibitor furegrelate (250 or 500µg; i.c.v.), or TXA2 receptor antagonist SQ-29548 (8 or 16µg; i.c.v.) was carried out 15min before AA (150µg; i.c.v.) injection. While indomethacin completely prevented the pressor and bradycardic responses to AA, furegrelate and SQ-29548 attenuated these effects in part in awake normotensive rats. In conclusion, these findings suggest that the pressor and bradycardic cardiovascular effects of centrally injected AA are dependent on COX activity being totally central and the TXA2 signaling pathway being subsequently central, at least in part.

Polyunsaturated fatty acids reduce Fatty Acid Synthase and Hydroxy-Methyl-Glutaryl CoA-Reductase gene expression and promote apoptosis in HepG2 cell line

Backgroundn-3 and n-6 polyunsaturated fatty acids (PUFAs) are the two major classes of PUFAs encountered in the diet, and both classes of fatty acids are required for normal human health. Moreover, PUFAs have effects on diverse pathological processes impacting chronic disease, such as cardiovascular and immune disease, neurological disease, and cancer.AimTo investigate the effects of eicosapentaenoic acid (EPA) and arachidonic acid (ARA) on the proliferation and apoptosis of human hepatoma cell line HepG2 after exposure to increasing concentrations of EPA or ARA for 48 h. Moreover, in the same cells the gene expression of Fatty Acid Synthase (FAS) and 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase (HMG-CoAR) was also investigated.MethodCell growth and apoptosis were assayed by MTT and ELISA test, respectively after cell exposure to increasing concentrations of EPA and ARA. Reverse-transcription and real-time PCR was used to detect FAS and HMG-CoAR mRNA levels in treated cells.ResultsOur findings show that EPA inhibits HepG2 cell growth in a dose-dependent manner, starting from 25 μM (P < 0.01, one-way ANOVA test and Dunnett's post test) and exerts a statistically significant pro-apoptotic effect already at 1 μM of EPA. Higher doses of ARA were need to obtain a statistically significant inhibition of cell proliferation and a pro-apoptotic effect in these cells (100 μM, P < 0.01, one-way ANOVA test and Dunnett's post test). Moreover, a down-regulation of FAS and HMG-CoAR gene expression was observed after EPA and ARA treatment in HepG2 cells, starting at 10 μM (P < 0.05, one-way ANOVA test and Dunnett's post test).ConclusionOur results demonstrate that EPA and ARA inhibit HepG2 cell proliferation and induce apoptosis. The down-regulation of FAS and HMG-CoAR gene expression by EPA and ARA might be one of the mechanisms for the anti-proliferative properties of PUFAs in an in vitro model of hepatocellular carcinoma.

Allometric scaling of dietary linoleic acid on changes in tissue arachidonic acid using human equivalent diets in mice

BackgroundIt is hypothesized that dietary linoleic acid (LA) promotes chronic and acute diseases in humans by enriching tissues with arachidonic acid (AA), its downstream metabolite, and dietary studies with rodents have been useful for validation. However, levels of LA in research diets of rodents, as published in the literature, are notoriously erratic making interspecies comparisons unreliable. Therefore, the ability to extrapolate the biological effects of dietary LA from experimental rodents to humans necessitates an allometric scaling model that is rooted within a human equivalent context.MethodsTo determine the physiological response of dietary LA on tissue AA, a mathematical model for extrapolating nutrients based on energy was used, as opposed to differences in body weight. C57BL/6J mice were divided into 9 groups fed a background diet equivalent to that of the US diet (% energy) with supplemental doses of LA or AA. Changes in the phospholipid fatty acid compositions were monitored in plasma and erythrocytes and compared to data from humans supplemented with equivalent doses of LA or AA.ResultsIncreasing dietary LA had little effect on tissue AA, while supplementing diets with AA significantly increased tissue AA levels, importantly recapitulating results from human trials.ConclusionsThus, interspecies comparisons for dietary LA between rodents and humans can be achieved when rodents are provided human equivalent doses based on differences in metabolic activity as defined by energy consumption.