Dietary n-3 and n-6 PUFA Enhance DHA Incorporation in Retinal Phospholipids Without Affecting PGE<Subscript>1</Subscript> and PGE<Subscript>2</Subscript> Levels

The purpose of this study was to determine whether dietary n-3 and n-6 PUFA may affect retinal PUFA composition and PGE₁ and PGE₂ production. Male Wistar rats were fed for 3 months with diets containing: (1) 10% eicosapentaenoic acid (EPA) and 7% docosahexaenoic acid (DHA), or (2) 10% γ-linolenic acid (GLA), or (3) 10% EPA, 7% DHA and 10% GLA, or (4) a balanced diet deprived of EPA, DHA, and GLA. The fatty acid composition of retinal phospholipids was determined by gas chromatography. Prostaglandin production was measured by enzyme immunoassay. When compared to rats fed the control diet, the retinal levels of DHA were increased in rats fed both diets enriched with n-3 PUFA (EPA + DHA and EPA + DHA + GLA diets) and decreased in those supplemented with n-6 PUFA only (GLA diet). The diet enriched with both n-6 and n-3 PUFA resulted in the greatest increase in retinal DHA. The levels of PGE₁ and PGE₂ were significantly increased in retinal homogenates of rats fed with the GLA-rich diet when compared with those of animals fed the control diet. These higher PGE₁ and PGE₂ levels were not observed in animals fed with EPA + DHA + GLA. In summary, GLA added to EPA + DHA resulted in the highest retinal DHA content but without increasing retinal PGE₂ as seen in animals supplemented with GLA only.     

Modulation of phorbol ester-associated events in epidermal cells by linoleate and arachidonate

To elucidate the events elicited by the skin tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA), which are modulated by linoleic acid (LA) and arachidonic acid (AA), the activity of these fatty acids in cultured mouse epidermal cells was compared. Approximately 94% of either exogenous radiolabelled fatty acid was incorporated into the total phospholipid pool over 15 h. The relative distribution among the phospholipid classes differed, however, such that approximately 70% of phospholipid-associated [¹⁴C]-LA was found in phosphatidylcholine, compared to approximately 30% for [¹⁴C]AA. Phosphatidylethanolamine and phosphatidylinositol/phosphatidylserine contained 17 and 13% of the phospholipid [¹⁴C]LA, and 34 and 30% of [¹⁴C]AA, respectively. Prostaglandin (PG) E₂ production was low but similar in unstimulated cultures prelabelled with either [¹⁴C]LA or [¹⁴C]AA. However, in cultures treated with TPA (1.6 μM), [¹⁴C]AA-prelabelling resulted in approximately three times the amount of [¹⁴C]PGE₂ compared with cultures prelabelled with [¹⁴C]LA. Cultured cells were found to contain significant δ6 desaturase activity, which may enable conversion of LA to AA, and thus may account for the observed PGE₂ production from [¹⁴C]LA treated cells. AA-Supplemented (1.6 μM) cultures supported approximately twice the induction of ornithine decarboxylase activity by TPA compared with cultures treated with 1.8 μM LA. Activation of partially purified protein kinase C was similar for either fatty acid tested over a 10–300 μM dose range. Overall, the results suggest that LA does not have the same biological activity as AA with regard to several TPA-associated events known to be important in skin tumor promotion. This reduced biological activity of LA may be partly responsible for the known inhibition of mouse skin tumor promotion by high dietary levels of LA [Leyton, J., Lee, M.L., Locniskar, M.F., Belury, M.A., Slaga, T.J., Bechtel, D., and Fischer, S.M. (1991)Cancer Res. 51, 907–915].     

Conjugated linoleic acid suppresses triglyceride accumulation and induces apoptosis in 3T3-L1 preadipocytes

Four sets of experiments were conducted to examine the influence of conjugated linoleic acid (CLA) isomers during proliferation and differentiation of cultures of 3T3-L1 preadipocytes using physiological culturing conditions. Cultures treated with either albumin [bovine serum albumin (BSA) vehicle] or linoleic acid (LA) served as controls. For the proliferation study (Expt. 1), cells were cultured in media containing a crude mixture of CLA isomers or pure LA at 0, 10, 50, or 200 μM for 4 d. Preadipocyte proliferation (cell number, ³H-thymidine incorporation into DNA) decreased as the level of CLA increased in the cultures. In contrast, LA had no impact on DNA synthesis. In Experiment 2a, postconfluent cultures were grown in media containing a crude mixture of CLA isomers or LA at 0, 10, 50, or 200 μM for the next 6 d. Postconfluent cultures supplemented with 50–200 μM CLA had less triglyceride (TG) and were smaller in size than cultures supplemented with similar amounts of LA. In Experiment 2b, postconfluent cultures supplemented with 200 μM of a crude mixture of CLA isomers or LA were harvested on days 1, 3, 6, or 9. Differences in TG content of cultures supplemented with 200 μM CLA compared to control and LA-supplemented cultures became apparent after 3 d of culture. Experiments 3a and 3b examined whether the fatty acid vehicle (BSA vs. ethanol) or the vitamin E status (±0.2 mM α-tocopherol) of the cultures altered CLA’s impact on preadipocyte TG content. In Experiment 3a, ethanol-treated cultures had more TG than non-ethanol-treated cultures regardless of the fatty acid treatment. In Experiment 3b, cultures treated with 100 μM of either a crude mixture of CLA or the trans-10, cis-12 CLA isomer without supplemental vitamin E for 6 d had less TG than CLA-treated cultures containing vitamin E. In Experiment 4, postconfluent cultures were grown in media containing 100 μM LA or either a crude mixture of CLA isomers or the trans-10, cis-12 CLA isomer for 24–96 h to assess CLA’s influence on the cell cycle and indices of apoptosis. Cultures treated with 100 μM CLA for 24–96 h had more apoptotic cells than BSA- or LA-treated cultures. Furthermore, cultures treated for 48 h with CLA had fewer cells in the S-phase than control cultures. The effects of the trans-10,cis-12 CLA isomer were more pronounced than those of the crude mixture of CLA isomers. These data suggest that CLA may exert its antiobesity effects by inhibiting proliferation, attenuating TG content, and/or inducing apoptosis in (pre)adipocytes.     

The effect of linoleic,arachidonic and eicosapentaenoic acid supplementation on prostacyclin production in rats

We examined the effect of dietary supplementation of linoleic acid (LA), arachidonic acid (AA) or eicosapentaenoic acid (EPA) to rats fed a diet low in linoleic acid onin vitro andin vivo production of prostacyclin. Male Sprague Dawley rats were fed a high-fat diet (50% energy as fat, 1.5% linoleic acid) for two weeks. Three of the groups were then supplemented orally with either 90 mg/d of LA, AA or EPA, all as the ethyl esters, for a further two weeks while remaining on the high-fat diet. Forty-eight hour urine samples were collected at the end of the second and fourth weeks.In vivo prostacyclin production was determined by a stable isotope dilution, gas chromatography/mass spectrometry assay for the major urinary metabolite of prostacyclins (2,3-dinor-6-keto-PGF_1α or PGI₂-M and Δ¹⁷-2-3-dinor-6-keto-PGF_1α or PGI₃-M).In vitro prostacyclin production was determined by radioimmunoassay of the stable metabolite (6-keto-PGF_1α) following incubation of arterial tissue. Oral supplementation with AA resulted in a rise in plasma and aorta 20∶4n−6, and increasedin vitro prostacyclin and urinary PGI₂-M production. EPA supplementation resulted in a rise in plasma and aorta 20∶5n−3 and 22∶5n−3, and a decline in plasma 20∶4n−6, but not in the aorta. In the EPA-supplemented group, thein vitro prostacyclin and the urinary PGI₃-M increased, but urinary PGI₂-M decreased. The increase inin vitro prostacyclin production in the EPA-supplemented rats was unexpected and without obvious explanation. Supplementation with LA had minimal effect on fatty acid composition of plasma or aorta and caused no change in prostacyclin production with either method. Thein vivo measure of prostacyclin production was positively correlated with aorta AA levels, and negatively correlated with aorta levels of EPA. There was a significant positive correlation between thein vitro production of prostacyclin and thein vivo production (as measured by the urinary prostacyclin metabolite level), despite the differences observed in the EPA-fed group. There was a high inter-animal variability in prostacyclin production using either method. These results indicate that dietary AA stimulates and dietary EPA reducesin vivo PGI₂ production in the rat. An equivalent amount of dietary LA was without effect.     

Eicosapentaenoic and arachidonic acid: Comparison of metabolism and activity in murine epidermal cells

The biological activity, including metabolism and modulation of ornithine decarboxylase activity and DNA synthesis, of arachidonic acid (AA) and eicosapentaenoic acid (EPA) were compared in epidermal cells from SENCAR mice. Radiolabelled AA and EPA were found to be similarly incorporated into and released from membrane phospholipids of unstimulated cultures. However, when cells were stimulated with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA), the release of AA was significantly higher than the release of EPA. The extent of metabolism of AA and EPA to prostaglandins was determined in both freeze-thawed cell preparations and in viable cultured cells. In the freeze-thawed preparations, use of AA as a substrate resulted in significantly more PGF than when EPA was used as the substrate. However, more PGE₃ was formed than PGE₂. PGD levels were the same for either fatty acid precursor. Prostaglandin production was also determined in viable cultured cells since other influences such as phospholipase A₂ activity can modify prostaglandin production. Control cultures prelabelled with either AA or EPA produced similar amounts of the respective PGF, PGE, and PGD. However, TPA-stimulated cultures produced significantly higher amounts of each prostaglandin in cultures prelabelled with AA compared to cells prelabelled with EPA. HETE or HEPE production was the same both for cultured cells prelabelled with AA or EPA and for homogenates from uncultured cells incubated directly with the radiolabelled fatty acids. TPA-induced ornithine decarboxylase (ODC) was significantly higher in AA-treated cultures compared to EPA-treated cultures. AA supports DNA synthesis to a greater extent than EPA, either alone or in the presence of TPA. These findings suggest that AA and EPA do not have equivalent biological activity in mouse epidermal cells.     

Inhibitory effect of conjugated linoleic acid on linoleic acid elongation in transformed yeast with human elongase

Conjugated linoleic acid (CLA; 18∶2) refers to a group of positional and geometric isomers derived from linoleic acid (LA; Δ9, 12–18∶2). Using a growing baker's yeast (Saccharomyces cerevisiae) transformed with human elongase gene, we examined the inhibitory effect of CLA at various concentrations (10, 25, 50, and 100 μM) on elongation of LA (25 μM) to eicosadienoic acid (EDA; Δ11,14–20∶2). Among four available individual CLA isomers, only c9,t11- and t10,c12-isomers inhibited elongation of LA to EDA. The extent of inhibition (ranging from 20 to 60%) was related to the concentration of CLA added to the medium. In the meantime, only these two isomers, when added at 50 μM to the media, were elongated to conjugated EDA (c11,t13- and t12,c14–20∶2) by the same recombinant elongase at the rate of 28 and 24%, respectively. The inhibitory effect of CLA on LA elongation is possibly due to competition between CLA isomers and LA for the recombinant elongase. Thus, results from this study and a previous study suggest that the biological effect of CLA is exerted through its inhibitory effect on Δ6-desaturation as well as elongation of LA which results in a decrease in long-chain n−6 fatty acids and consequently the eicosanoid synthesis.     

Enhancement of Polyunsaturated Fatty Acid Production by Selenium Treatment in Polyunsaturated Fatty Acid-Producing Fungus

The effect of selenium (Se) treatment on the content of intracellular polyunsaturated fatty acids (PUFA) was investigated. Selenium treatment could effectively increase the yields of arachidonic acid (AA) and linoleic acid (LA). When AA-producing Diasporangium jonesianum recombinant strain D-A1 was cultured in a medium containing 90 μg mL⁻¹ Se, the growth of D. jonesianum recombinant strain D-A1 was constrained, but the content of AA in the total fatty acids reached 9.3%, 1.7-fold higher than the control. The content of LA was also increased from 25.9 to 37.7%. Under various culture conditions, Se treatment (90 μg mL⁻¹) was generally beneficial to the yields of LA and AA. However, there was no significant increase in the contents of eicosapentaenoic acid (EPA) and γ-linolenic acid. Therefore, the use of Se is advantageous for regulating the content of intracellular PUFA in D. jonesianum recombinant strain D-A1, and could be good enhancer in the yields of AA and LA.     

Platelet and aorta arachidonic and eicosapentaenoic acid levels andin vitro eicosanoid production in rats fed high-fat diets

There is a significant interest in the interrelationship between long-chain n-3, and n-6 fatty acids due to their ability to modulate eicosanoid production. In general, the intake of arachidonic acid (AA) results in enhanced eicosanoid production, whereas n-3 polyunsaturated fatty acids (PUFA) decrease the production of eicosanoids from AA. The purpose of this study was to investigate whether the effects of dietary AA on eicosanoid production in the rat were correlated with the AA and EPA levels in platelets and aorta (eicosanoid-producing tissues). Four groups of male Sprague-Dawley rats were fed a highfat diet enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (approximately 100 mg/day of EPA+DHA) for 24 d. During the last 10 d, the four groups were orally supplemented with 0,30,60, and 90 mg/day of ethyl arachidonate. A further group of rats was fed a control diet (without longchain n-3 PUFA) for 24 d.In vitro aorta prostacyclin (PGI₂) production, serum thromboxane A₂ (TxA₂) production and plasma, and platelet and aorta phospholipid (PL) fatty acids were measured. Enriching the diet with n-3 PUFA resulted in significant reductions in tissue AA levels and an increase in the n-3 PUFA, particularly EPA. On this diet, the AA to EPA ratio was 1:1 in platelet PL, and it was 2:1 in the aorta PL. There were significant decreases in thein vitro PGI₂ and TxA₂ production compared with the control animals. The inclusion of AA in the diet resulted in marked increases in AA levels in the platelet and aorta PL with corresponding decreases in EPA. The lowest dose of AA (30 mg/rat) reversed the effects of 100 mg/day of n-3 PUFA on AA levels in platelet and aortic PL and onin vitro aorta PGI₂ and serum TxA₂ production. The dietary AA caused a differential (twofold) increase in TxA₂ relative to PGI₂ for all three levels of AA supplementation. There were greater changes in the levels of AA and/or EPA in platelet PL compared with the aorta PL, which might have accounted for the differential effects of these PUFA on thromboxane production compared with PGI₂ production in this study.     

Conjugated linoleic acids alter bone fatty acid composition and reduce ex vivo prostaglandin E2 biosynthesis in rats fed n-6 or n-3 fatty acids

This study evaluated the effects of conjugated linoleic acids (CLA) on tissue fatty acid composition and ex vivo prostaglandin E₂ (PGE₂) production in rats given diets varying in n-6 and n-3 fatty acids. Four groups of rats were given a basal semipurified diet (AIN-93G) containing 70 g/kg of added fat for 42 d. The fat treatments were formulated to contain CLA (0 vs. 10 g/kg of diet) and n-6 (soybean oil having an n-6/n-3 ratio of 7.3) and n-3 fatty acids (menhaden oil+safflower oil having an n-6/n-3 ratio of 1.8) in different ratios in a 2×2 factorial design. Fatty acids in liver, serum, muscle, heart, brain, spleen, and bone (cortical, marrow, and periosteum) were analyzed by capillary gas-liquid chromatography. The various dietary lipid treatments did not affect growth; however, CLA improved feed efficiency. The CLA isomers were found in all rat tissues analyzed although their concentrations varied. Dietary CLA decreased the concentrations of 16∶1n−7, 18∶1, total monounsaturates and n−6 fatty acids, but increased the concentrations of n−3 fatty acids (22∶5n−3 and 22∶6n−3), and saturates in the tissues analyzed. Ex vivo PGE₂ production in bone organ culture was decreased by n−3 fatty acids and CLA. We speculate that CLA reduced the concentration of 18∶1 fatty acids by inhibiting liver Δ9-desaturase activity. The fact that CLA lowered ex vivo PGE₂ production in bone organ culture suggests that these conjugated fatty acids have the potential to influence bone formation and resorption.     

Docosahexaenoic acid and other dietary polyunsaturated fatty acids suppress leukotriene synthesis by mouse peritoneal macrophages

The efficacy of individual ω-t-3 polyunsaturated fatty acids (PUFA) in altering eicosanoid synthesis in peritoneal macrophages was studied by feeding mice for 10 days a diet containing 2 wt% fat, which included 0.5 wt% ethyl esters of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or linolenic acid (LNA). Upon stimulation with calcium ionophore A23187, macrophages from these animals produced significantly lower amounts of leukotriene C₄, leukotriene B₄ and 12-hydroxyeicosatetraenoic acid, prostaglandin E₂ and 6-keto prostaglandin F_1α compared with those obtained from animals on the diets containing olive oil or safflower oil. The decrease in leukotriene synthesis was similar in the animals fed DHA, EPA or LNA diets. This depression of eicosanoids by DHA and EPA was associated with decreased levels of arachidonic acid (AA); however, LA that altered eicosanoids did not have the same effect on AA levels.     

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.     

Δ6 Desaturase mRNA Abundance in HepG2 Cells Is Suppressed by Unsaturated Fatty Acids

The effect of unsaturated fatty acids on the abundance of Δ6 desaturase (D6D) mRNA and the fatty acid composition of HepG2 cell membranes was examined. Supplementation of HepG2 cells with oleic acid (18:1n-9, OA), linoleic acid (18:2n-6, LA), α-linolenic acid (18:3n-3, ALA), arachidonic acid (20:4n-6, AA) or eicosapentaenoic acid (20:5n-3, EPA) reduced D6D mRNA abundance by 39 ± 6.6, 40 ± 2.2, 31 ± 5.2, 55 ± 4.8, and 52 ± 5.0%, respectively, compared with control cells. Despite the reduction in D6D mRNA abundance, the level of D6D conversion products (20:3n-9, EPA and AA) in OA, ALA and LA supplemented cells, respectively, was elevated above that in control cells. Our results suggest that although unsaturated fatty acids decrease the abundance of D6D mRNA by as much as 50%, the conversion of polyunsaturated fatty acids and accumulation of long chain polyunsaturated fatty acids (LCPUFA) in HepG2 cell phospholipids continues to occur.     

The significance of polyunsaturated fatty acids in cutaneous biology

The skin epidermis displays a highly active metabolism of polyunsaturated fatty acids (PUFA). Dietary deficiency of linoleic acid (LA) and 18-carbon (n-6) PUFA results in characteristic scaly skin disorder and excessive epidermal water loss. Arachidonic acid, a 20-carbon (n-6) PUFA is metabolized via the cyclooxygenase pathway into predominantly prostaglandin E₂ (PGE₂) PGF_2α′ and PGD₂ and via the lipoxygenase pathway into predominantly 15-hydroxyeicosatetraenoic acid (15-HETE). The prostaglandins modulate normal skin physiological processes at low concentrations and inflammatory reactions at high concentrations. Similarly, the very active epidermal 15-lipoxygenase transforms dihomogammalinolenic acid (DGLA) into 15-hydroxy eicosatrienoic acid (15-HETrE), eicosapentaenoic acid (EPA) into 15-hydroxyeicosapentaenoic acid (15-HEPE) and docosahexaenoic acid (DHA) into 17-hydroxydocosahexaenoic acid (17-HDoHE), respectively. These monohydroxy acids exhibit anti-inflammatory properties. In contrast, the 18-carbon (n-6) PUFA is transformed into 13-hydroxy-9,11-octadecadienoic acid (13-HODE), which exerts antiproliferative properties in the tissue. Thus the supplementation of diets with appropriate purified vegetable oils and/or fish oil may generate local cutaneous anti-inflammatory metabolites which could serve as a less toxicin vivo monotherapy or as adjuncts to standard therapeutic regimens for the management of skin inflammaory disorders.     

Comparison of the effects of dietary alpha-linolenic,stearidonic, and eicosapentaenoic acids on production of inflammatory mediators in mice

The effects of dietary stearidonic acid (18∶4n−3) on inflammatory mediator release in whole blood and splenocytes was investigated in Balb/c mice, and the effects were compared with those of two other n−3 PUFA: α-linolenic acid (18∶3n−3) and EPA (20∶5n−3). TAG mixtures containing 10% of 18∶4n−3, 18∶3n−3, or 20∶5n−3 as the respective sole n−3 PUFA were enzymatically synthesized. Diets containing synthesized TAG mixtures were fed to Balb/c mice for 3 wk. The release of prostaglandin E₂ (PGE₂) and tumor necrosis factor (TNF) were measured in whole blood and splenocytes stimulated with lipopolysaccharide. In whole blood, the production of INF was suppressed by all dietary n−3 PUFA (18∶3n−3, 18∶4n−3, and 20∶5n−3) as compared with the control diet, which contained TAG prepared from safflower oil. PGE₂ production was not significantly changed. Differences among the n−3 PUFA (18∶3n−3), 18∶4n−3, and 20∶5n−3) were not observed. In splenocytes, PGE₂ production was suppressed by dietary n−3 PUFA, but TNF production was not. GC analysis of plasma and splenocyte FA profiles showed an increase in the levels of 20∶4n−3, 20∶5n−3, and 22∶6n−3 in mice fed the diet containing 18∶4n−3.     

Modulation of prostaglandin H synthase activity by conjugated linoleic acid (CLA) and specific CLA isomers

Conjugated linoleic acid (CLA) has been shown to inhibit tumorigenesis in animal models and is cytostatic to numerous cell lines in vitro. However, the mechanism of action is unknown. In the current study, we determined the effects of CLA and specific isomers of CLA on the rate of oxygenation of arachidonic acid by prostaglandin H synthase (PGHS) in ram seminal vesicle microsomes. The enzyme was incubated with 0.1 to 100 μM CLA or specific isomers of CLA for 2 min prior to the addition of 44 to 176 μM arachidonate. The isomers tested were 9(E),11(E) CLA; 9(Z),11(E) CLA; 9(Z),11(Z) CLA, and 10(E),12(Z) CLA. For a positive inhibitor control, flurbiprofen was used at 0.75 to 2.50 μM. Enzyme activity was assessed by measuring the rate of oxygen consumption. Inclusion of CLA or specific isomers of CLA in the incubation mixtures inhibits PGHS. The efficacy differs for each isomer, with the 9(Z),11(E) CLA isomer being the most effective and the 9(Z),11(Z) CLA isomer being the least effective inhibitor among the four CLA isomers tested. The K _i values obtained by Dixon replots range from 18.7 μM for the most effective isomer, 9(Z),11(Z) CLA, to 105.3 μM for the least effective isomer, 9(Z),11(Z) CLA. The K _i value for flurbiprofen with ram seminal vesicle microsomes was 0.33 μM. As the concentration of arachidonate was increased, the CLA-dependent inhibition of PGHS decreased, suggesting competitive inhibition. The results of this study demonstrate the potential of CLA and specific isomers of CLA to modulate prostaglandin biosynthesis.     

Protective action of CLA against oxidative inactivation of paraoxonase 1, an antioxidant enzyme

The effect of CLA on paraoxonase 1 (PON1), one of the antioxidant proteins associated with HDL, was investigated for its protective action against oxidative inactivation as well as its stabilization activity. When cis-9 (c9),trans-11 (t11)-CLA and t10,c12-CLA were examined for their protective activity against ascorbate/Cu²⁻-induced inactivation of PON1 in the presence of Ca²⁺, two CLA isomers exhibited a remarkable protection (E _max, 71–74%) in a concentration-dependent manner (50% effective concentration, 3–4 μM), characterized by a saturation pattern. Such a protective action was also reproduced with oleic acid, but not linoleic acid. Rather, linoleic acid antagonized the protective action of CLA isomers in a noncompetitive fashion. Additionally, the two CLA isomers also protected PON1 from oxidative inactivation by H₂O₂ or cumene hydroperoxide. The concentration-dependent protective action of CLA against various oxidative inactivation systems suggests that the protective action of CLA isomers may be mediated through their selective binding to a specific binding site in a PON1 molecule. Separately, the inactivation of PON1 by p-hydroxymercuribenzoate (PHMB), a modifier of the cysteine residue, was also prevented by CLA isomers, suggesting the possible existence of the cysteine residue in the binding site of CLA. The c9,t11-CLA isomer seems to be somewhat more effective than t10,c12-CLA in protecting against the inactivation of PON1 by either peroxides or PHMB, in contrast to the similar efficacy of these two CLA isomers in preventing ascorbate/Cu²⁺-induced inactivation of PON1. Separately, CLA isomers successfully stabilized PON1, but not linoleic acid. These data suggest that the two CLA isomers may play a beneficial role in protecting PON1 from oxidative inactivation as well as in its stabilization.     

Polyunsaturated fatty acids regulate cytokine and prostaglandin E2 production by respiratory cells in response to mast cell mediators

A protective association between breastfeeding and the development of bronchial asthma has been demonstrated. However, a mechanism remains unclear. FA present in human milk but rare in infant formula have been associated with marked immunological modulation as well as some indications of protection from asthma development. We examined the effect of in vitro manipulation of membrane phospholipid on the production of cytokines and prostaglandin (PG)E₂ by respiratory epithelial cells (A549) in response to stimulation by mast cell mediators of allergic disease [histamine, tumor necrosis factor (TNF)-α, interleukin (IL)-4 and IL-5]. DHA and CLA significantly decreased the production of IL-8 in response to stimulation by TNF-α [2907±970 (DHA) and 6471±1203 (CLA) vs. 12,287±2309 (control) pg/ml; P≤0.05, mean±SEM], whereas both EPA and DHA reduced histamine-stimulated RANTES (regulation on activation, T cell-expressed and-secreted) production [2314±861 (EPA) and 877±326 (DHA) vs. 8526±1118 (control) pg/ml; P≤0.03]. PGE₂ released in response to histamine was decreased by n−3 [1305±399 (α-linolenic acid), 406±73 (EPA), and 265±32 (DHA) vs. 9324±3672 (control) pg/mL; P≤0.05] and increased by n−6 [18,843±4439 (arachidonic acid) vs. 9324±3672 (control) pg/mL; P=0.02], with CLA producing a decrease of the same magnitude as DHA [553±126 (CLA) vs. 9324±3672 (control) pg/mL; P=0.03]. This study demonstrates the potential for immunological manipulation of the respiratory epithelium by FA in situ during allergic responses and suggests that further investigation into FA intervention in infants via human milk or supplemented infant formula, to prevent the development of allergic disease, may be worthwhile.     

Metabolism of dietary α-linolenic acid vs. eicosapentaenoic acid in rat immune cell phospholipids during endotoxemia

Short-term (i.e., 3 d) continuous enteral feeding of diets containing eicosapentaenoic (EPA) and γ-linolenic (GLA) polyunsaturated fatty acids (PUFA) to endotoxemic rats reduces the levels of arachidonic acid (AA) and linoleic acid (LA) in alveolar macrophage (AM) and liver Kupffer and endothelial (K&E) cell phospholipids with attendant decreases in prostaglandin formation by these cells in vitro. Diets that contain α-linolenic acid (LNA) as a substrate for endogenous formation of EPA may not be as effective in facilitating these immune cell modifications given the limited activity of Δ6 desaturase. In the present study we compared the effectiveness of an LNA-enriched diet vs. an (EPA+GLA)-enriched diet to displace phospholipid AA from AM and liver K&E cells in vivo in endotoxemic rats fed enterally for 3 or 6 d. We determined the fatty acid composition of AM and K&E cell phospholipids by gas chromatography. We found that AM and K&E cells from rats that had received the EPA+GLA diet for 3 d had significantly (P<0.001) higher mole percentage of EPA and the GLA metabolite, dihomoGLA, than corresponding cells from rats given the LNA diet or a control diet enriched with LA. Rats given the LNA diet had relatively low levels of stearidonic acid, EPA and other n−3 PUFA, while rats given the LA diet had low levels of GLA and dihomoGLA. We conclude that diets enriched with LNA or LA may not be as effective as those enriched with FPA+GLA for purposes of fostering incorporation of EPA or dihomoGLA into and displacement of AA from macrophage phospholipids under pathophysiologic conditions commonly found in acutely septic patients.     

Modulation of respiratory syncytial virus-induced prostaglandin E<Subscript>2</Subscript> production by n−3 long-chain polyunsaturated fatty acids in human respiratory epithelium

Infection with respiratory syncytial virus (RSV) results in substantial infant morbidity and has been associated with the subsequent development of childhood asthma. Inflammatory mediators produced by both the epithelium and tissue leukocytes during RSV infection stimulate the release of chemotactic factors by the respiratory epithelium and the subsequent influx of inflammatory cells, predominantly neutrophils. We investigated the production of inflammatory mediators [prostaglandin E₂ (PGE₂), interleukin (IL)-1β, tumor necrosis factor α] and chemokines [IL-8, RANTES (regulation on activation, normal T cell expressed and secreted)] by alveolar epithelial cells in response to RSV infection. Infection of a human alveolar epithelial transformed cell line (A549 cells) with live RSV substantially increased production of PGE₂, IL-8, and RANTES. By altering cell membrane FA through incorporation of the long-chain PUFA (LCPUFA) arachidonic acid, EPA, and DHA, we were subsequently able to significantly modulate PGE₂ production by the infected epithelium. Because of the dynamic nature of the effects of PGE₂ on lung function, regulation of this prostaglandin during RSV infection by n−3 LCPUFA has the potential to significantly alter the disease process.