Polyunsaturated fatty acids increase the sensitivity of 36B10 rat astrocytoma cells to radiation-induced cell kill

Polyunsaturated fatty acids (PUFA) such as γ-linolenic acid (GLA, 18:3n-6), eicosapentaenoic acid (EPA, 20: 5n-3), and docosahexaenoic acid (DHA, 22:6n-3) have been shown to be cytotoxic to tumor cells. The objective of this work was to study the effect of PUFA on the radiation response of a 36B10 rat astrocytoma cell line. Supplementation of the astrocytoma cells with 15–45 μM GLA, EPA, or DHA produced marked changes in the fatty acid profiles of their phospholipids and neutral lipids. The methylene bridge index of these lipids increased significantly. These PUFA also exerted cytotoxic effects, as determined using the clonogenic cell survival assay. While GLA and DHA produced a moderate cell-killing effect, EPA was extremely cytotoxic, especially at a concentration of 45 μM. The monounsaturated oleic acid (OA, 18:1n-9) did not affect cell survival. Further, all three PUFA, and particularly GLA, increased the radiation-induced cell kill; OA did not enhance the effect of radiation. α-Tocopherol acetate blocked the enhanced radiation sensitivity of GLA- and DHA-supplemented cells. In conclusion, GLA, EPA, and DHA supplementation prior to, during, and after irradiation can enhance the radiation-induced cytotoxicity of rat astrocytoma cells. GLA and DHA supplementation post-irradiation also enhanced the radiation response of the 36B10 cells. Because GLA maximally increases the radioresponsiveness of a rat astrocytoma, this PUFA might prove useful in increasing the therapeutic efficacy of radiation in the treatment of certain gliomas.     

Cell Proliferation and Long Chain Polyunsaturated Fatty Acid Metabolism in a Cell Line From Southern Bluefin Tuna (Thunnus maccoyii)

Southern bluefin tuna (SBT, Thunnus maccoyii) aquaculture is a highly valuable industry, but research on these fish is hampered by strict catch quotas and the limited success of captive breeding. To address these limitations, we have developed a SBT cell line (SBT-E1) and here we report on fatty acid metabolism in this cell line. The SBT-E1 cells proliferated well in standard Leibovitz’s L-15 cell culture medium containing fetal bovine serum (FBS) as the source of fatty acids. Decreasing the FBS concentration decreased the cell proliferation. Addition of the C₁₈ polyunsaturated fatty acids (PUFA) α-linolenic acid (ALA, 18:3n-3) or linoleic acid (LNA, 18:2n-6) to the cell culture medium had little effect on the proliferation of the cells, whereas addition of the long-chain PUFA (LC-PUFA) arachidonic acid (ARA, 20:4n-6), eicosapentaenoic acid (EPA, 20:5n-3) or docosahexaenoic acid (DHA, 22:6n-3) significantly reduced the proliferation of the cells, especially at higher concentrations and especially for DHA. Addition of vitamin E to the culture medium overcame this effect, suggesting that it was due to oxidative stress. The fatty acid profiles of the total lipid from the cells reflected those of the respective culture media with little evidence for desaturation or elongation of any of the fatty acids. The only exceptions were EPA and ARA, which showed substantial elongation to 22:5n-3 and 22:4n-6, respectively, and DHA, which was significantly enriched in the cells compared with the culture medium. The results are discussed in light of the dietary PUFA requirements of SBT in the wild and in aquaculture.     

Suppression of growth in a leukemic T cell line by n−3 and n−6 polyunsaturated fatty acids

Proliferation in a lekemic T cell line (Jurkat) was suppressed in a dose dependent manner by n−6 and n−3 polyunsaturated fatty acids (PUFA) added to the culture medium. At high concentrations, PUFA have a cytotoxic effect on Jurkat cells. The inhibitory effect of the PUFA was not due to production of prostaglandins, and lipid peroxidation was only partly responsible. In addition to production of peroxides and aldehydes, lipid peroxidation also reduced the plasmalogen levels in these cells. The antioxidant α-tocopherol blocked lipid peroxidation and restored the plasmalogen levels to normal. α-Tocopherol did not totally restore cell proliferation although the MDA-like products in these cultures (supplemented with PUFA) were reduced to control level. Cultures supplemented with n−6 PUFA seemed to respond better to α-tocopherol than n−3 PUFA. This suggests that n−6 PUFA may exert their growth inhibitory effect predominantly via lipid peroxidation while different mechanisms might be operating for the n−3 PUFA.     

Polyunsaturated fatty acids exert antiarrhythmic actions as free acids rather than in phospholipids

Previous studies have shown that exogenous free n-3 polyunsaturated fatty acids (PUFA) can prevent tachyarrhythmias caused by specific agents in isolated cardiac myocytes. However, the question as to whether incorporation of the n-3 PUFA into membrane phospholipids has the same immediate protective effects remained to be answered. To answer this question, we increased the content of n-3 PUFA in the phospholipids of cultured neonatal rat myocytes by growing them 2–3 d in a culture to which eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA) in 15 μM concentration was added. Analysis of the fatty acid composition of membrane phospholipids revealed a significantly higher level of EPA and DHA (from 0.2 to 7.6% and from 1.2 to 6.5%) in cells supplemented with EPA or DHA, respectively. The responses of the myocytes grown in normal media or in media enriched with the PUFA to arrhythmogenic agents were examined after free fatty acids were removed from the medium and the cells. The arrhythmogenic agents used were the β-adrenergic agonist isoproterenol or an elevated extracellular concentration of calcium. The results showed that there was no significant difference in the induction of tachyarrhythmias by isoproterenol or by elevated [Ca²⁺]_o in cells grown in media enriched with PUFA, as compared with cells grown in normal media in the absence of the free PUFA. Under the conditions of this study, only the unesterified PUFA were able to protect the cardiomyocytes against induced arrhythmias. There was no antiarrhythmic effect due to an increased fraction of EPA or DHA in membrane phospholipids.     

Season and Cooking May Alter Fatty Acids Profile of Polar Lipids from Blue-Back Fish

Polar lipids (PoL) represent a new promising dietary approach in the prevention and treatment of many human diseases, due to their potential nutritional value and unique biophysical properties. This study investigates the effects of catching season and oven baking on the fatty acid profiles (FAP) of PoL in four species of blue-back fish widely present in the North Adriatic Sea: anchovy (Engraulis encrasicholus), sardine (Sardina pilchardus), sprat (Sprattus sprattus), and horse mackerel (Trachurus trachurus). PoL levels (427–652 mg/100 g flesh) varied among the four species, with no significant seasonal variations within species. FAP of raw fillets were particularly high in polyunsaturated fatty acid (PUFA), especially docosahexaenoic acid (DHA) and EPA; total PUFA was constant in all species throughout the year, while long-chain n-3 polyunsaturated fatty acid (n-3 PUFA) rose in spring (except in sprat), especially due to the contribution of DHA. The FAP response for PoL to oven baking was species-specific and, among n-3 PUFA, DHA exhibited the greatest heat resistance; the influence of oven baking on FAP was found to be correlated with the catching season, especially for anchovy and sardine, while sprat PoL were not affected by cooking processes. The four species analyzed in this study presented very low n-6/n-3 fatty acid ratios and highly favorable nutritional indices, emphasizing their PoL qualities and promoting their role in increasing human n-3 PUFA intake. The four species can be considered as superior sources of n-3 PUFA and can be employed as supplements in functional food manufacturing and in pharmaceutical and cosmetic industries.     

Synergistic In Vitro Anticancer Toxicity of Pulsed Electric Fields and Glutathione

Despite continuous advancement in skin cancer therapy, the disease is still fatal in many patients, demonstrating the need to improve existing therapies, such as electrochemotherapy (ECT). ECT can be applied in the palliative or curative setting and is based on the application of pulsed electric fields (PEF), which by themselves exerts none to low cancer toxicity but become potently toxic when combined with low-dosed chemotherapeutics such as bleomycin and cisplatin. Albeit their favorable side-effect profiles, not all patients respond to standard ECT, and some responders experience tumor recurrence. To identify potential adjuvant or alternative agents to standard electrochemotherapy, we explored the possibility of combining PEF with a physiological compound, glutathione (GSH), to amplify anticancer toxicity. GSH is an endogenous antioxidant and is available as a dietary supplement. Surprisingly, neither GSH nor PEF mono treatment but GSH + PEF combination treatment exerted strong cytotoxic effects and declined metabolic activity in four skin cancer cell lines in vitro. The potential applicability to other tumor cells was verified by corroborating results in two leukemia cell lines. Strikingly, GSH + PEF treatment did not immediately increase intracellular GSH levels, while levels 24 h following treatment were enhanced. Similar tendencies were made for intracellular reactive oxygen species (ROS) levels, while extracellular ROS increased following combination treatment. ROS levels and the degree of cytotoxicity could be partially reversed by pre-incubating cells with the NADPH-oxidase (NOX) inhibitor diphenyleneiodonium (DPI) and the H₂O₂-degrading enzyme catalase. Collectively, our findings suggest a promising new “endogenous” drug to be combined with PEF for future anticancer research approaches.     

α‐Linolenic acid metabolism in adult humans: the effects of gender and age on conversion to longer‐chain polyunsaturated fatty acids

This review summarises and evaluates current knowledge of α‐linolenic acid (αLNA) metabolism in adult humans. The principal biological role of αLNA appears to be as a precursor for the synthesis of longer‐chain n‐3 polyunsaturated fatty acids (PUFA). Stable isotope tracer studies indicate that conversion of αLNA to eicosapentaenoic acid (EPA) occurs but is limited in men and that further transformation to docosahexaenoic acid (DHA) is very low. A lower proportion of αLNA is used for β‐oxidation in women compared with men, while the fractional conversion to the longer‐chain n‐3 PUFA is greater, possibly due to the regulatory effects of oestrogen. Increasing αLNA intake for a period of weeks results in an increase in the proportion of EPA in plasma lipids, circulating cells and breast milk, but there is no increase in DHA, which may even decline in some pools at high αLNA intakes. Overall, αLNA appears to be a limited source of longer‐chain n‐3 PUFA in man, and so adequate intakes of preformed long‐chain n‐3 PUFA, in particular DHA, may be important for maintaining optimal tissue function. The capacity to up‐regulate αLNA transformation in women may be important for meeting the demands of the foetus and neonate for DHA.     

Accretion of Dietary Docosahexaenoic Acid in Mouse Tissues Did Not Differ between Its Purified Phospholipid and Triacylglycerol Forms

Recent studies suggest that dietary krill oil leads to higher omega-3 polyunsaturated fatty acids (n-3 PUFA) tissue accretion compared to fish oil because the former is rich in n-3 PUFA esterified as phospholipids (PL), while n-3 PUFA in fish oil are primarily esterified as triacylglycerols (TAG). Tissue accretion of the same dietary concentrations of PL- and TAG-docosahexaenoic acid (22:6n-3) (DHA) has not been compared and was the focus of this study. Mice (n = 12/group) were fed either a control diet or one of six DHA (1%, 2%, or 4%) as PL-DHA or TAG-DHA diets for 4 weeks. Compared with the control, DHA concentration in liver, adipose tissue (AT), heart, and eye, but not brain, were significantly higher in mice consuming either PL- or TAG-DHA, but there was no difference in DHA concentration in all tissues between the PL- or TAG-DHA forms. Consumption of PL- and TAG-DHA at all concentrations significantly elevated eicosapentaenoic acid (20:5n-3) (EPA) in all tissues when compared with the control group, while docoshexapentaenoic acid (22:5n-6) (DPA) was significantly higher in all tissues except for the eye and heart. Both DHA forms lowered total omega-6 polyunsaturated fatty acids (n-6 PUFA) in all tissues and total monounsaturated fatty acids (MUFA) in the liver and AT; total saturated fatty acid (SFA) were lowered in the liver but elevated in the AT. An increase in the DHA dose, independent of DHA forms, significantly lowered n-6 PUFA and significantly elevated n-3 PUFA concentration in all tissues. Our results do not support the claim that the PL form of n-3 PUFA leads to higher n-3 PUFA tissue accretion than their TAG form.     

Protective Effects of EPA and Deleterious Effects of DHA on eNOS Activity in Ea hy 926 Cultured with Lysophosphatidylcholine

Oxidized low density lipoprotein (Ox-LDL) is a well-established risk factor in atherosclerosis and lysophosphatidylcholine (LysoPtdCho) is considered to be one of the major atherogenic component of Ox-LDL. The purpose of this work was to investigate the effects of two membrane n-3 long chain polyunsaturated fatty acids (n-3 PUFAs), EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) compared to n-6 PUFA, ARA (arachidonic acid), on the activation of endothelial NO synthase (eNOS) by histamine in Ea hy 926 endothelial cells incubated during 24 h in the presence or the absence of LysoPtdCho. DHA (50 μM) produced a ROS induction in cells and aggravated the LysoPtdCho-induced oxidative stress. It did not modify the basal eNOS activity but impaired the stimulation of eNOS induced by histamine and was unable to correct the deleterious effect of LysoPtdCho on histamine-stimulated eNOS activity or phosphorylation of Ser 1177. In contrast, EPA (90 μM) did not modify the ROS level produced in the presence or absence of LysoPtdCho or basal eNOS activity and the stimulating effect of histamine on eNOS. However, it diminished the deleterious effect of LysoPtdCho as well as on the histamine-stimulated eNOS activity on the phosphorylation on Ser 1177 of eNOS. The beneficial effect of EPA but not DHA on endothelial eNOS activity in Ea hy 926 could be also partially due to a slight decrease in membrane DHA content in EPA-treated cells. Consequently, the equilibrium between NO generated by eNOS and ROS due to oxidative stress could explain, in part, the beneficial effect of EPA on the development of cardiovascular diseases. By contrast ARA an n-6 PUFA was devoid of any effect on ROS generation or eNOS activity in the basal state or after histamine-induced stimulation. In vivo experiments should be undertaken to confirm these results.     

Dietary Crude Lecithin Increases Systemic Availability of Dietary Docosahexaenoic Acid with Combined Intake in Rats

Crude lecithin, a mixture of mainly phospholipids, potentially helps to increase the systemic availability of dietary omega‐3 polyunsaturated fatty acids (n‐3 PUFA), such as docosahexaenoic acid (DHA). Nevertheless, no clear data exist on the effects of prolonged combined dietary supplementation of DHA and lecithin on RBC and plasma PUFA levels. In the current experiments, levels of DHA and choline, two dietary ingredients that enhance neuronal membrane formation and function, were determined in plasma and red blood cells (RBC) from rats after dietary supplementation of DHA‐containing oils with and without concomitant dietary supplementation of crude lecithin for 2–3 weeks. The aim was to provide experimental evidence for the hypothesized additive effects of dietary lecithin (not containing any DHA) on top of dietary DHA on PUFA levels in plasma and RBC. Dietary supplementation of DHA‐containing oils, either as vegetable algae oil or as fish oil, increased DHA, eicosapentaenoic acid (EPA), and total n‐3 PUFA, and decreased total omega‐6 PUFA levels in plasma and RBC, while dietary lecithin supplementation alone did not affect these levels. However, combined dietary supplementation of DHA and lecithin increased the changes induced by DHA supplementation alone. Animals receiving a lecithin‐containing diet also had a higher plasma free choline concentration as compared to controls. In conclusion, dietary DHA‐containing oils and crude lecithin have synergistic effects on increasing plasma and RBC n‐3 PUFA levels, including DHA and EPA. By increasing the systemic availability of dietary DHA, dietary lecithin may increase the efficacy of DHA supplementation when their intake is combined.     

Utilization of Aminoguanidine Prevents Cytotoxic Effects of Semen

Studies of human semen in cell or tissue culture are hampered by the high cytotoxic activity of this body fluid. The components responsible for the cell damaging activity of semen are amine oxidases, which convert abundant polyamines, such as spermine or spermidine in seminal plasma into toxic intermediates. Amine oxidases are naturally present at low concentrations in seminal plasma and at high concentrations in fetal calf serum, a commonly used cell culture supplement. Here, we show that, in the presence of fetal calf serum, seminal plasma, as well as the polyamines spermine and spermidine, are highly cytotoxic to immortalized cells, primary blood mononuclear cells, and vaginal tissue. Thus, experiments investigating the effect of polyamines and seminal plasma on cellular functions should be performed with great caution, considering the confounding cytotoxic effects. The addition of the amine oxidase inhibitor aminoguanidine to fetal calf serum and/or the utilization of serum-free medium greatly reduced this serum-induced cytotoxicity of polyamines and seminal plasma in cell lines, primary cells, and tissues and, thus, should be implemented in all future studies analyzing the role of polyamines and semen on cellular functions.     

Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis,and Enhances the Chemosensitivity of Cancer Cells

The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells’ molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies.     

The Influence of Polyunsaturated Fatty Acids on the Phospholipase D Isoforms Trafficking and Activity in Mast Cells

The impact of polyunsaturated fatty acid (PUFA) supplementation on phospholipase D (PLD) trafficking and activity in mast cells was investigated. The enrichment of mast cells with different PUFA including α-linolenic acid (LNA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) or arachidonic acid (AA) revealed a PUFA-mediated modulation of the mastoparan-stimulated PLD trafficking and activity. All PUFA examined, except AA, prevented the migration of the PLD1 to the plasma membrane. For PLD2 no PUFA effects on trafficking could be observed. Moreover, PUFA supplementation resulted in an increase of mastoparan-stimulated total PLD activity, which correlated with the number of double bonds of the supplemented fatty acids. To investigate, which PLD isoform was affected by PUFA, stimulated mast cells were supplemented with DHA or AA in the presence of specific PLD-isoform inhibitors. It was found that both DHA and AA diminished the inhibition of PLD activity in the presence of a PLD1 inhibitor. By contrast, only AA diminished the inhibition of PLD activity in the presence of a PLD2 inhibitor. Thus, PUFA modulate the trafficking and activity of PLD isoforms in mast cells differently. This may, in part, account for the immunomodulatory effect of unsaturated fatty acids and contributes to our understanding of the modulation of mast cell activity by PUFA.     

Profile of polyunsaturated fatty acids produced by Thraustochytrium sp. KK17-3

More than 300 strains of microorganisms producing polyunsaturated fatty acids (PUFA) were newly isolated from coastal seawater in the Seto Inland Sea and around Iriomote Island, Japan, by the baiting method. The profiles of PUFA from docosahexaenoic acid (DHA)-producing strains could be classified into four types. A strain, named KK17-3, was chosen for further study owing to its high DHA content (52.1% of total fatty acid) and wide range of PUFA (76.1%) including arachidonic, eicosapentaenoic, and docosapentaenoic acids as well as DHA. Glucose and tryptone were the optimal carbon and nitrogen sources, respectively, in a medium with salinity at 75% that of seawater. The PUFA contents in polar lipids (22.1% of total lipid), in which the DHA content was 39.3%, were higher than those in neutral lipids and glycolipids. Molecular phylogenetic analysis of 18S rRNA gene sequences showed KK17-3 to be a thraustochytrid. It also was observed to possess a life cycle composed of vegetative cells without successive bipartition, zoosporangium, and zoospore stage. Classification by the chemotaxonomic criterion based on PUFA compositions also supported this assignment.     

Effects of n−3 and n−6 fatty acids on tumor necrosis factor cytotoxicity in WEHI fibrosarcoma cells

Modulation by fatty acids of the cytotoxic effect of recombinant tumor necrosis factor alpha (TNF) toward WEHI 164 mouse fibrosarcoma cells has been examined. Preincubating the highly TNF-sensitive WEHI clone 13 cells for 44 hr with 50 μmol/L of 20∶5n−3, 22∶6n−3, 18∶3n−6, 20∶3n−6 or 20∶4n−6 reduced cell survival 22 hr after challenge with TNF (40 ng/L) by 65%, 72%, 60%, 98% and 85%, respectively. In comparison, 18∶3n−3, 18∶2n−6 and 18∶1n−9 had only negligible effects on TNF-induced toxicity. Different extent of fatty acid incorporation into cell total phospholipids or triglycerides could not explain the observed effects on TNF cytotoxicity, and the enhanced cytotoxicity could therefore not be explained merely by an increased unsaturation of the cell membranes. In addition to the fatty acid supplied, preincubation with 18∶2n−6, 18∶3n−6 or 18∶3n−3 also enriched the cells with 20∶2n−6, 20∶3n−6 and 20∶3n−3, respectively, most likely due to chain elongation. The results suggest that the WEHI cells have a low Δ6 desaturase activity, and that n−6 and n−3 acids must have at least 3 or 4 double bonds, respectively, to enhance TNF cytotoxicity in WEHI cells. Dexamethasone partly inhibited TNF-induced cytotoxicity, while cyclooxygenase, thromboxane synthetase or lipoxygenase inhibitors had no or negligible effects. The antioxidant butylated hydroxyanisole (BHA) completely inhibited TNF-induced cytotoxicity, while the structurally and functionally similar antioxidant butylated hydroxy-toluene had no such effect, indicating that BHA does not block TNF cytotoxicity through its antioxidant effect. The results suggest that TNF cytotoxicity involves, directly or indirectly, metabolism of long-chain polyun-saturated fatty acids, and we speculate that fatty acid metabolites are involved.     

Specific modifications of phosphatidylinositol and nonesterified fatty acid fractions in cultured porcine cardiomyocytes supplemented with n-3 polyunsaturated fatty acids

Mechanisms for the antiarrhythmic effect of n−3 polyunsaturated fatty acids (PUFA) are currently being investigated using isolated cardiac myocytes. It is still not known whether the incorporation of n−3 PUFA into membrane phospholipids is a prerequisite for its protective action or if n−3 PUFA exert antiarrhythmic effects in their nonesterified form as demonstrated by recent studies. Adult porcine cardiomyocytes were grown in media supplemented with arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). After 24 h, analysis of total lipids showed that the myocytes were enriched with the respective fatty acids compared to control cells. Large proportions of all three fatty acids supplemented (69% AA, 72% DHA, and 66% EPA) remained unesterified. Fatty acid analysis of total phospholipids (PL) revealed that the incorporation of EPA and DHA, though small, was significantly different (P<0.05) from that of the control cells. The PL fraction was further separated into phosphatidylinositol (Pl), phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine to study the pattern of incorporation of the fatty acids in these fractions. It became apparent that EPA and DHA were selectively incorporated into the Pl fraction. This study demonstrates that in adult porcine cardiomyocytes, the n−3 PUFA supplementation selectively modulates two important lipid fractions, nonesterified fatty acid and Pl, which were implicated in the mechanisms of prevention of cardiac arrhythmias.     

Matrix Extension Validation of AOCS Ce 2c-11 for Omega-3 Polyunsaturated Fatty Acids in Conventional Foods and Dietary Supplements Containing Added Marine Oil

Marine oils are commonly added to conventional foods and dietary supplements to enhance their contents of omega-3 polyunsaturated fatty acids (PUFA), including eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), which have been associated with numerous potential health benefits. This study compared American Oil Chemists’ Society (AOCS) Official Methods Ce 2b-11 and Ce 2c-11 for determining EPA and DHA in foods and dietary supplements and found that AOCS Ce 2c-11 produces significantly higher analyzed values, which could be attributed to a more comprehensive breakdown of the sample matrix and derivatization of fatty acids. Our subsequent food matrix extension validation of AOCS Ce 2c-11 demonstrated that the method produces true, accurate, sensitive, and precise determinations of EPA, DHA, and total omega-3 PUFA in foods and dietary supplements containing added marine oil, including those formulated with emulsified and microencapsulated oils. The method detection limits for EPA and DHA were 0.012 ± 0.002 and 0.011 ± 0.003 mg g⁻¹, respectively (means ± SD). The analyzed contents of EPA (1.26–386 mg serving⁻¹), DHA (1.37–563 mg serving⁻¹), and total omega-3 PUFA (2.69–1270 mg serving⁻¹) were reported for 27 conventional food and dietary supplement products. Eighteen products declared contents of DHA, EPA + DHA, or total omega-3 PUFA on product labels, and the analyzed contents of those fatty acids varied from 95 to 162% of label declarations for all but two of the products.     

Butylated hydroxyanisole inhibits tumor necrosis factor-induced cytotoxicity and arachidonic acid release

The mechanisms by which the antioxidant butylated hydroxyanisole (BHA) inhibits recombinant tumor necrosis factor alpha (rTNF-α)-induced cytotoxicity have been studied in WEHI 164 clone 13 (WEHI) and L929 fibrosarcoma cells. When BHA was added simultaneously with rTNF-α, it completely inhibited rTNF-α cytotoxicity in the WEHI and L929 cells. BHA also inhibited the toxicity when added 2 h after rTNF-α in WEHI cells, suggesting that BHA inhibits some late intracellular event(s) in rTNF-α cytotoxicity. Pretreating WEHI cells with BHA for 4 h did not decrease the binding of rTNF-α to its receptors as measured using flow cytometry. BHA inhibited rTNF-α toxicity in the presence of actinomycin D and cycloheximide, indicating that neither mRNA nor protein synthesis is necessary for the BHA effect. The antioxidant butylated hydroxytoluene (BHT) and indomethacin did not inhibit the rTNF-α-induced cytotoxicity nor the rTNF-α-induced release of [³H]arachidonic acid. By comparison, BHA completely inhibited the rTNF-α-induced release of arachidonic acid, suggesting that BHA somehow inhibits rTNF-α-induced activation of phospholipase(s). In WEHI cells, rTNF-α increased the level of protein-associated thiobarbituric acid reactive substances (TBARS) dose-dependently. BHA, but not BHT, blocked rTNF-α-induced cytotoxicity and rTNF-α-induced accumulation of protein-associated TBARS, suggesting that rTNF-α cytotoxicity is correlated with protein-associated TBARS. In conclusion, the results suggest that BHA blocks some post receptor event in rTNF-α-induced cytotoxicity, and that activation of phospholipase(s) coupled with the enzymatic formation of specific oxidized lipids could be a pivotal event in rTNF-α-induced cytotoxicity.     

Incorporation of polyunsaturated fatty acids into CT-26, a transplantable murine colonic adenocarcinoma

Previous studies in our laboratory have shown that marine oils, with high levels of eicosapentaenoic (EPA, 20∶5n−3) and docosahexaenoic acids (DHA, 22∶6n−3), inhibit the growth of CT-26, a murine colon carcinoma cell line, when implanted into the colons of male BALB/c mice. Anin vitro model was developed to study the incorporation of polyunsaturated fatty acids (PUFA) into CT-26 cells in culture. PUFA-induced changes in the phospholipid fatty acid composition and the affinity with which different fatty acids enter the various phospholipid species and subspecies were examined. We found that supplementation of cultured CT-26 cells with either 50 μM linoleic acid (LIN, 18∶2n−6), arachidonic acid (AA, 20∶4n−6), EPA, or DHA significantly alters the fatty acid composition of CT-26 cells. Incorporation of these fatty acids resulted in decreased levels of monounsaturated fatty acids, while EPA and DHA also resulted in lower levels of AA. While significant elongation of both AA and EPA occurred, LIN remained relatively unmodified. Incorporation of radiolabeled fatty acids into different phospholipid species varied significantly. LIN was incorporated predominantly into phosphatidylcholine and had a much lower affinity for the ethanolamine phospholipids. DHA had a higher affinity for plasmenylethanolamine (1-O-alk-1′-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) than the other fatty acids, while EPA had the highest affinity for phosphatidylethanol-amine (1,2-diacyl-sn-glycero-3-phosphoethanolamine). These results demonstrate that,in vitro, significant differences are seen between the various PUFA in CT-26 cells with respect to metabolism and distribution, and these may help to explain differences observed with respect to their effects on tumor growth and metastasis in the transplantable model.