The intestinal mucosa as a target for dietary polyunsaturated fatty acids.

Several studies have reported beneficial effects of dietary polyunsaturated fatty acids (PUFA) on various aspects of both human and animal health, and particular reference has been made to their effects on systemic immune responses. Both immune stimulation and immune suppression have been reported, with the outcome dependent on the type of PUFA, the target cell, as well as the immune competence of the cells before exposure. The systemic and the mucosal immune systems are discrete entities, which have evolved specific approaches in the defense of the host. The latter comprises several interconnected tissues, which communicate with one another through the action of soluble mediators and the trafficking of cellular components. After the oral mucosa, the intestinal epithelium and its associated gut-associated lymphoid tissue are the primary targets of dietary components. Absorption of dietary PUFA and its incorporation into intestinal tissues has been well studied, but the consequences of these events in relation to local immune responses have received little attention. This article describes some of the immune mechanisms operating at this barrier and, where possible, pinpoints areas for which a modulatory role for PUFA has already been demonstrated. Although not an exhaustive treatise of the subject, it is hoped that this review will foster research into the specific interaction between dietary PUFA and cell populations comprising the intestinal barrier.

Short-term fish oil supplementation improved innate immunity, but increased ex vivo oxidation of LDL in man--a pilot study.

BACKGROUND: Fish and fish oils are rich in the two long-chain polyunsaturated fatty acids (LCPUFAs) eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). The n-3 LCPUFAs have been reported to have beneficial effects on cardiovascular functions, but their role in relation to immune functions is still controversial. AIM OF THE STUDY: The objectives of this study were to determine the effects of supplementation with fish oil on immune cell functions in human subjects. We have also assessed the effects on plasma lipids, antioxidant status and susceptibility of low-density lipoproteins (LDL) to oxidative stress. The antioxidant status was determined by measuring plasma vitamin C, tocopherols and carotenoids in plasma and LDL, and superoxide dismutase (SOD) in red blood cells. DESIGN: For 30 days, 10 volunteers ingested 25 g/d of either fish oil, providing n-3 LCPUFAs (7.5 g), or high-oleic sunflower oil, providing monounsaturated fatty acids mainly as oleic acid (22 g). The oils contained similar profiles of tocopherols. At day 0 and day 30, blood samples were drawn by venipuncture for plasma lipid and antioxidant analyses and lipoprotein isolation, and for isolation and functional tests of mononuclear cells and granulocytes. Fatty acid profiles of im mune cells and LDL were also determined. RESULTS: Fish oil supplementation resulted in an accumulation of n-3 LCPUFAs (EPA, DHA) in LDL and immune cells. The phagocytic activity, a measure of immune cell activity, was increased in both groups. Whereas the plasma and LDL antioxidant status do not appear to be affected by fish oil supplementation, an increased susceptibility of LDL to oxidation was observed in these healthy volunteers. CONCLUSIONS: The optimal amounts of n-3 fatty acids required to modulate immune functions remain to be established. In addition, adequate levels of antioxidant protection need to be provided during fish oil supplementation.

Urinary isoprostane excretion is not confounded by the lipid content of the diet.

This study aims to determine if isoprostanes accurately reflect in vivo lipid peroxidation or whether they are influenced by the lipid content of the diet. Isoprostanes were measured in urine of healthy subjects under different conditions of lipid intake and under conditions of oxidative stress (fasting). We found that isoprostanes were not influenced by the lipid content of the diet: the urinary level remained constant over 24 h as well as over 4 consecutive days when switching from high to low lipid intake. Urinary isoprostane excretion was increased by 40% following a 24 h fast. We concluded that urinary isoprostane excretion reflects endogenous lipid peroxidation in vivo.

Effects of a fish-oil and vegetable-oil formula on aggregation and ethanolamine-containing lysophospholipid generation in activated human platelets and on leukotriene production in stimulated neutrophils.

The effects of consuming a liquid formula containing either fish oil enriched in omega-3 fatty acids or vegetable oil enriched in oleic acid was evaluated in 20 male subjects randomly allocated into two groups over a 42-d period. A decrease in collagen-induced aggregation by using washed platelet suspensions was found in both groups after nutritional supplementation. A considerable rise in omega-3 and a decrease in omega-6 fatty acids occurred in the platelet phospholipid with fish-oil consumption. The degree of eicosapentaenoic acid (EPA, 20:5n-3) enrichment (fish-oil group) was dramatically greater in the ether-containing plasmenylethanolamine (13.5 mol% of fatty acids; mol% of fatty acids = moles per 100 moles of total fatty acids) than in phosphatidylethanolamine (2.8 mol%) or phosphatidylcholine (2.9 mol%). Neither treatment significantly influenced the agonist-induced accumulation of lysoplasmenylethanolamine as derived via phospholipase A2 hydrolysis of plasmenylethanolamine. HPLC measurements of eicosanoid production in A23187-stimulated neutrophils revealed a considerable decrease in the formation of arachidonic acid-derived leukotriene B4 (LTB4), by 41%, and 5-HETE (5-hydroxyeicosatetraenoic acid), by 30%, in the fish-oil group along with the appearance of the corresponding EPA-derived products [LTB5 and 5-HEPE (5-hydroxyeicosapentaenoic acid)]. No such alterations in the formation of lipoxygenase products were found with the vegetable oil treatment.

The cleavage of plasmenylethanolamine by phospholipase A2 appears to be mediated by the low affinity binding site of the TxA2/PGH2 receptor in U46619-stimulated human platelets.

Two TxA2/PGH2 receptor binding sites linked to different effector systems have recently been identified. Since plasmenylethanolamine represents the major phospholipid reservoir of arachidonic acid (AA) in resting human platelets, we assessed the differential role of these binding sites on plasmenylethanolamine hydrolysis by phospholipase A2 activity upon platelet activation by determining the generation of the corresponding [3H]lysoplasmenylethanolamine. Ethanolamine-containing phospholipids in platelets were pre-labelled with [3H]ethanolamine prior to platelet stimulation with U46619 (1 microM), a TxA2 mimetic, in the presence or absence of S-145, an antagonist of the low affinity TxA2/PGH2 receptor. Labelled platelets were also treated with the TxA2/PGH2 receptor antagonist, GR32191B, prior to washing (which blocks the low affinity site of the receptor) and subsequent stimulation. The above conditions provided for blockage of platelet aggregation but not shape change with U46619. The rise in [3H]lysoplasmenylethanolamine accumulation (170% of unstimulated controls) with U46619 as the agonist was inhibited in platelets pre-treated with S-145 and in platelets washed from GR32191B. Similar findings were also obtained for [3H]lysophosphatidylethanolamine accumulation. The present results indicate that the TxA2-dependent activation of plasmenylethanolamine cleavage by phospholipase A2 in intact human platelets is predominantly linked to the low affinity site of the TxA2/PGH2 receptor and may be important for platelet aggregation but not shape change.

Correlation between platelet aggregation and dephosphorylation of a 68 kDa protein revealed through the use of putative PKC inhibitors.

The efficacy of two structurally and functionally unrelated protein kinase C (PKC) inhibitors, chelerythrine and calphostin C, was assessed in intact human platelets by studying platelet aggregation in response to stimulation with phorbol 12-myristate 13-acetate (PMA) or the thromboxane-A2 mimetic, U46619. Surprisingly, both inhibitors increased aggregation in response to PMA, but decreased aggregation in response to U46619. To further explore this phenomenon, gel electrophoresis of 32P-labelled proteins from PMA- or U46619-stimulated platelets in the presence and absence of the two putative PKC inhibitors was performed. Although neither chelerythrine nor calphostin C proved to be effective PKC inhibitors in intact human platelets, a strong correlation between the dephosphorylation of a 68 kDa protein and the rate of platelet aggregation was observed. From these results, the indiscriminate use of PKC inhibitors in whole platelets is questioned and attention is drawn to the role of protein dephosphorylation in platelet activation. The 68 kDa protein was the major phosphorylated substrate in resting platelets. Okadaic acid increased phosphorylation of this band, indicating active phosphate group turnover under resting conditions.

Eicosanoid/thromboxane A2-independent and -dependent generation of lysoplasmenylethanolamine via phospholipase A2 in collagen-stimulated human platelets.

Collagen-induced human platelet stimulation is dependent on the release of arachidonic acid (AA) from membrane phospholipid and the formation of thromboxane A2 (TxA2) for TxA2-induced platelet activation. Since plasmenylethanolamine represents the single major phospholipid reservoir of AA in resting human platelets, we assessed its hydrolysis via phospholipase A2 upon platelet stimulation with low levels of collagen by determining the generation of [3H]lysoplasmenylethanolamine via eicosanoid/TxA2-independent and -dependent processes. Ethanolamine phospholipids in platelets were prelabelled with [3H]ethanolamine before stimulation with either collagen or the TxA2 mimetic U46619, in the presence or absence of BW755C, a dual inhibitor of the cyclooxygenase/lipoxygenase activities, or GR32191B, a TxA2-receptor antagonist. Collagen stimulation promoted a marked generation of [3H]lysoplasmenylethanolamine, which was only moderately decreased when TxA2 synthesis or TxA2 receptors were blocked by BW755C or GR32191B respectively. The moderate rise in [3H]lysoplasmenylethanolamine formation with U46619 as the agonist was only slightly affected by BW755C and blocked by GR32191B. Evidence for eicosanoid/TxA2-independent and -dependent generation of [3H]lysophosphatidylethanolamine was also obtained. A significant quantitative loss of AA from plasmenylethanolamine was also demonstrated in collagen-stimulated platelets. The present findings indicate the activation of plasmenylethanolamine cleavage via phospholipase A2 in collagen-stimulated human platelets, which, to a considerable extent, does not depend on eicosanoid/TxA2 synthesis. This may represent an important source of releasable AA for TxA2 generation and the promotion of further liberation of AA and phospholipid-mediated signalling pathways.

Lipid composition and peroxide levels of mucosal cells in the rat large intestine in relation to dietary fat.

To examine whether dietary fat alters membrane lipid composition and peroxidation of polyunsaturated fatty acids in "non-proliferative" and "proliferative" cells in the large intestine, Sprague-Dawley rats were fed diets providing a polyunsaturated-to-saturated fatty acid ratio of 1.2 or 0.3 at a high or low level of fat intake for a 25-day period. Cell populations were isolated and the effect of dietary fat on membrane polyunsaturated fatty acid content and peroxide levels was determined. Neither fat level nor fatty acid composition of diet influenced total cholesterol, total phospholipids, and percentage of phospholipid classes in membrane phospholipids. Feeding the high fat and/or high polyunsaturated-to-saturated fatty acid ratio diet increased polyunsaturated fatty acid content of mucosal cell phospholipids. Increase in polyunsaturated fatty acid content was paralleled by a decrease in the monounsaturated fatty acid content of mucosal cell phospholipids. Membrane content of total saturated fatty acids was not significantly affected by diet. Variation in phospholipid fatty acid composition between "non-proliferative" and "proliferative" cells was observed. Lipid peroxide levels in mucosal cell lipid fractions were altered by dietary fat treatment. Animals fed high fat diets, compared to groups fed low fat diets, exhibited higher membrane peroxide levels when results are expressed as nmol/mg protein. Higher peroxide levels were observed in mucosal cells for rats fed high polyunsaturated-to-saturated fatty acid ratio diets when results were expressed per nmol of phospholipid. It is concluded that changes in fat level and fatty acid composition of the diet alters the mucosal cell membrane lipid composition in the rat large intestine and influences susceptibility of mucosal cell lipid to peroxidation. Further research is required to delineate which dietary factors--fat level, polyunsaturated-to-saturated fatty acid ratio, or both--have a primary influence on the degree of lipid peroxidation.