The effects of dietary n-3 polyunsaturated fatty acids on neutrophils.

The studies of dietary fish oil supplementation in healthy volunteers demonstrate a significant increase in neutrophil EPA content, a concomitant reduction in neutrophil AA content, and suppression of neutrophil LTB4 synthesis by supplementation with dietary fish oil containing approximately 3-4 g EPA daily for a minimum of 4 weeks. Suppression of neutrophil chemotactic responsiveness to LTB4 and FMLP was observed after dietary n-3 PUFA supplementation at these levels. Dietary EPA is more active than DHA in eliciting these effects in human neutrophils. Dietary n-3 PUFA supplementation inhibits neutrophil chemotaxis to these ligands through the inhibition of the signal transduction pathway between the receptor and phospholipase C, as demonstrated by the inhibition of chemotaxin-stimulated IP3 formation, in the absence of an effect on the number or affinity of the respective chemotaxin receptors. In patients with RA, dietary supplementation with n-3 PUFA resulted in decreased AA content of cellular lipids, with an augmented EPA content and decreased LTB4 generation by neutrophils. Dietary supplementation with n-3 PUFA also resulted in augmentation of depressed neutrophil chemotaxis to LTB4 and FMLP. Preliminary findings suggest that the decreased responsiveness to chemotaxins of neutrophils from RA patients is due to down-regulation of chemotaxin receptor number, resulting in decreased signaling via chemotaxin receptors. Dietary fish oil PUFA partially reversed the down-regulation of the chemotaxin receptor of neutrophils of RA patients, but had a lesser effect on chemotaxin receptor signaling and function, probably due to a post-receptor inhibition induced by fish oil PUFA, as was previously observed in healthy controls. Several small clinical trials have each suggested that dietary supplementation with n-3 PUFA resulted in modest improvements in disease activity. Meta-analysis of these studies confirms statistically significant improvements in tender joint count and morning stiffness after 3 months of dietary fish oil supplementation in patients with RA. Dietary supplementation with gamma-linolenic acid-rich oils also inhibits neutrophil LTB4 formation, has other anti-inflammatory and immunosuppressive effects, and shows promise of therapeutic efficacy in RA.

Extensive reversible brain magnetic resonance lesions in a patient with HELLP syndrome.

A severe form of toxemia of pregnancy with microangiopathic hemolytic anemia, elevated liver enzymes, and low platelets has been called the HELLP syndrome. A patient with the HELLP syndrome developed a severe, reversible encephalopathy. Brain computed tomography and magnetic resonance imaging showed abnormalities consistent with edema limited to the posterior circulation territory. The location of the lesions and their occurrence in the HELLP syndrome support suggestions that the vulnerability of posterior structures in eclamptic encephalopathy is due to a vascular susceptibility of the posterior circulation and that endothelial cell dysfunction plays an important role in the pathogenesis of eclamptic encephalopathy.

Novel, ligation-dependent PCR assay for detection of hepatitis C in serum.

A simple, sensitive, and specific ligation-dependent PCR (LD-PCR) method for the detection of hepatitis C virus (HCV) RNA in serum is described. The assay uses two DNA capture probes for RNA isolation and two DNA hemiprobes for subsequent PCR. Each capture probe has a 3' sequence complementary to the conserved 5' untranslated region of HCV RNA and a biotin moiety at the 5' end capable of interacting with streptavidin-coated paramagnetic beads. Each hemiprobe contains a sequence complementary to the 5' untranslated region in juxtaposition to one another and a common sequence for PCR primer binding. In guanidinium thiocyanate solutions, the capture probes and the hemiprobes form a hybrid with their target, and the hybrid can be isolated from serum by the binding of the capture probes to the paramagnetic beads in the presence of a magnetic field. The hemiprobes can then be linked to each other by incubation with T4 DNA ligase to form a full probe that serves as a template for a PCR. When serial 10-fold dilutions of synthetic HCV RNA (10(7) to 10 molecules) were tested, there was a good correlation between the amount of PCR product and the initial number of RNA molecules, with a sensitivity of 100 HCV RNA molecules per reaction. Twenty-four specimens that had been tested by either a branched DNA probe (bDNA) assay (13 specimens) or a reverse transcription PCR (RT-PCR) assay (11 specimens) were also analyzed by LD-PCR. The results showed a good correlation among LD-PCR, RT-PCR, and the bDNA assay. However, both LD-PCR and RT-PCR were more sensitive than the bDNA assay when the HCV titer was low.

Validation of a meta-analysis: the effects of fish oil in rheumatoid arthritis.

The purpose of this study was to validate the results of a meta-analysis showing the efficacy of fish oil in rheumatoid arthritis with the results of a re-analysis of the complete primary data set. A Medline search yielded seven published papers. Three additional trials were found by contacting authorities in the field. Inclusion criteria included (1) a double-blind, placebo-controlled study, (2) use of at least one of seven predetermined outcome measures, (3) results reported for both placebo and treatment groups at baseline and follow-up, (4) randomization, and (5) parallel or cross-over design. Papers were scored for quality. Demographic and outcomes variables were collected. For the re-analysis of the primary data, the same variables were abstracted for the 395 individual patients randomized. The meta-analysis demonstrated that dietary fish oil supplementation for 3 months significantly reduced tender joint count (rate difference [RD] [95% CI] = -2.9 [-3.8 to -2.1] [p = 0.001]) and morning stiffness (RD [95% CI] = -25.9 [-44.3 to -7.5] [p < 0.01]) as compared with heterogeneous dietary control oils. The re-analysis of the primary data confirmed a significant reduction in tender joint count (p = 0.001) and in morning stiffness (p < 0.02) in the parallel analysis that ignored interaction terms. The analyses that included an interaction term between site and treatment again confirmed a significant reduction in tender joint count. The results for morning stiffness were similar to the meta-analysis, but did not quite reach statistical significance (p = 0.052-0.083). The relative improvements in the other outcome variables did not reach statistical significance. Use of fish oil improved the number of tender joints and duration of morning stiffness at 3 months as analyzed by both meta- and mega-analysis. The fuller mega-analysis confirmed the results of the meta-analysis. The advantages of mega-analysis were as follows: (1) the ability to analyze the homogeneity of the patient populations, (2) the ability to make clinically sensible adjustments in the form of the comparison, and (3) the ability to examine subsets of the data.

Eicosanoids in rheumatoid arthritis.

Eicosanoids are potent mediators in the cellular microenvironment. Eicosanoids have different effects depending on tissue or organ, the polyunsaturated fatty acid content of the diet of the individual, and the net effect of local microenvironmental factors--as eicosanoids, cytokines, and hormones modulate each others' effects through a complex, multilevel network of interactions. In general, eicosanoids have significant net proinflammatory effects. In RA, the net proinflammatory effects of the prostanoids is underscored by the effectiveness of the cyclooxygenase antagonists (NSAIDs), and recent data indicate a proinflammatory effect of the leukotrienes. Changes in the dietary polyunsaturated fatty acid composition to increased intake of marine n-3 fatty acids and/or dihomogamma-linolenic acid may favorably modulate eicosanoid synthesis towards less inflammatory or antiinflammatory eicosanoids and may ameliorate disease activity in RA. Recent advances in the biochemistry and molecular biology of the eicosanoid receptors and the synthetic pathways of eicosanoids will provide opportunities for advances in the therapeutics for RA, including selective cyclooxygenase (PGHS-2) antagonists, selective eicosanoid receptor antagonists and agonists, and selective inhibitors of PGH2 isomerases and enzymes of the 5-lipoxygenase pathway.

Dietary omega-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils.

Earlier studies demonstrated that dietary omega-3 polyunsaturated fatty acid (PUFA) supplementation attenuates the chemotactic response of neutrophils and the generation of leukotriene (LT) B4 by neutrophils stimulated with calcium ionophore; however, the mechanisms and relationship of these effects were not examined. Neutrophils and monocytes from eight healthy individuals were examined before and after 3 and 10 wk of dietary supplementation with 20 g SuperEPA daily, which provides 9.4 g eicosapentaenoic acid (EPA) and 5 g docosahexaenoic acid. The maximal neutrophil chemotactic response to LTB4, assessed in Boyden microchambers, decreased by 69% after 3 wk and by 93% after 10 wk from prediet values. The formation of [3H]inositol tris-phosphate (IP3) by [3H]inositol-labeled neutrophils stimulated by LTB4 decreased by 71% after 3 wk (0.033 +/- 0.013% [3H] release, mean +/- SEM) and by 90% after 10 wk (0.011 +/- 0.011%) from predict values (0.114 +/- 0.030%) as quantitated by beta-scintillation counting after resolution on HPLC. LTB4-stimulated neutrophil chemotaxis and IP3 formation correlated significantly (P < 0.0001); each response correlated closely and negatively with the EPA content of the neutrophil phosphatidylinositol (PI) pool (P = 0.0003 and P = 0.0005, respectively). Neither the affinities and densities of the high and low affinity LTB4 receptors on neutrophils nor LTB4-mediated diglyceride formation changed appreciably during the study. Similar results were observed in neutrophils activated with platelet-activating factor (PAF). The summed formation of LTB4 plus LTB5 was selectively inhibited in calcium ionophore-stimulated neutrophils and was also inhibited in zymosan-stimulated neutrophils. The inhibition of the summed formation of LTB4 plus LTB5 in calcium ionophore-stimulated neutrophils and in zymosan-stimulated neutrophils did not correlate significantly with the EPA content of the PI pool. The data indicate that dietary omega-3 PUFA supplementation inhibits the autoamplification of the neutrophil inflammatory response by decreasing LTB4 formation through the inactivation of the LTA epoxide hydrolase and independently by inhibiting LTB4- (and PAF) stimulated chemotaxis by attenuating the formation of IP3 by the PI-selective phospholipase C. This is the initial demonstration that dietary omega-3 PUFA supplementation can suppress signal transduction at the level of the PI-specific phospholipase C in humans.

Dietary omega-3 fatty acids: effects on lipid mediators of inflammation and rheumatoid arthritis.

Dietary supplementation omega-3 polyunsaturated fatty acids may inhibit (at least partially) three pathways of the synthesis of lipid mediators of inflammation: the platelet-activating factor synthesis pathway, the cyclooxygenase pathway, and the 5-lipoxygenase pathway. In addition, selected cellular functions of polymorphonuclear neutrophils may be modulated by dietary fish oil. The exact mechanism of the effects of dietary supplementation with omega-3 poly-unsaturated fatty acids on these pathways is not completely elucidated; it is quite probable that the effects will vary with the duration, dose, and composition of the omega-3 polyunsaturated fatty acid preparation, with background medical therapy, and the presence of and degree of activity of the underlying inflammatory disease state.

Effects of dietary fish oil on leukocyte leukotriene and PAF generation and on neutrophil chemotaxis.

The studies of dietary fish oil supplementation in healthy volunteers demonstrate: (1) suppression of PMN LTB4 synthesis after a minimum of 4 weeks of dietary fish oil consumption at a level of 4-6 g omega 3 fatty acids daily; concomitant suppression of the other arachidonate-derived 5-lipoxygenase pathway products and decreased [3H]-arachidonic acid release may be observed under certain conditions, (2) suppression of PMN chemotactic responsiveness to LTB4 and FMLP, (3) delayed kinetics of inhibition of chemotaxis and AA metabolism relative to that of cellular lipid alteration, and (4) dietary EPA is more active than DHA in eliciting these effects. The effects of dietary EPA on monocyte function in healthy volunteers include: (1) suppression of LTB4 synthesis concomitantly with that of the other 5-lipoxygenase pathway products and decreased [3H]-arachidonic acid release, (2) suppression of PAF synthesis, and (3) delayed kinetics of inhibition of PAF generation and AA metabolism relative to that of cellular lipid alteration. The effects of dietary fish oil in RA patients include: (1) decreased arachidonate content of cellular lipids with an augmented EPA content, (2) decreased LTB4 generation by PMN as an isolated effect, indicating inhibition of the epoxide hydrolase enzyme. The decrease in LTB4 generation by PMN correlated with improvement of tender joint count in one study, (3) augmentation of depressed PMN chemotaxis to LTB4 and FMLP, and (4) suppression of monocyte PAF generation. From these studies one may conclude that: (1) omega 3 fatty acids are incorporated into leukocyte cellular phospholipids with a concomitant loss in arachidonic acid, (2) the incorporation of omega 3 fatty acids into leukocyte cellular lipids suppresses two pathways of inflammatory mediator synthesis: the 5-lipoxygenase and the PAF synthesis pathways, (3) receptor-mediated PMN functions are altered by dietary omega 3 fatty acid consumption, and (4) these functional changes may be delayed vis-a-vis changes in cellular lipid composition and may vary with the underlying disease states and/or background medication.

The effects of N-3 polyunsaturated fatty acids on the generation of platelet-activating factor-acether by human monocytes.

Human leukocytes generate platelet-activating factor (PAF-acether), a lipid mediator of inflammation, from membrane alkyl phospholipids through the release of arachidonic acid or other fatty acids at the 2-position and subsequent acetylation. Because it was previously demonstrated that fish oil fatty acids suppress human leukocyte arachidonic acid release and metabolism, separate experiments were conducted to investigate the effects of dietary fish oil supplementation and in vitro incubation with fish oil fatty acids on calcium ionophore-stimulated PAF-acether generation in human monocytes. In subjects on their regular diets, a 4-hr incubation of monocyte monolayers with an optimally effective concentration of arachidonic acid of 1 micrograms/ml resulted in a 64% increase of calcium ionophore-induced net PAF-acether generation from 7.75 +/- 0.78 ng/10(6) cells for untreated monolayers to 12.70 +/- 1.21 ng/10(6) cells (mean +/- SEM). Treatment of monolayers with eicosapentaenoic acid (EPA) at the optimal concentration of 1 micrograms/ml decreased net PAF-acether generation by 28%. However, treatment of monocyte monolayers with docosahexaenoic acid did not appreciably affect net PAF-acether generation. The changes in PAF-acether release with each fatty acid added in vitro paralleled those in total PAF-acether generation; the percentage PAF-acether release remained unaffected. Three weeks of dietary supplementation with 18 g MaxEPA daily, providing 3.2 g EPA did not affect the PAF-acether generation of calcium ionophore-stimulated human monocyte monolayers. However, 6 weeks of dietary supplementation resulted in a 47% decrease of net total PAF-acether generation and a concomitant 59% decline in net PAF-acether release; the percentage release of PAF-acether was not affected. Thus, whether added to the diet or introduced in vitro, fish oil-derived fatty acids suppress PAF-acether generation by human monocyte monolayers.

Effects of dietary supplementation with marine fish oil on leukocyte lipid mediator generation and function in rheumatoid arthritis.

Twelve patients with active rheumatoid arthritis supplemented their usual diet with 20 gm of Max-EPA fish oil, daily, for 6 weeks. Following this supplementation, the ratio of arachidonic acid to eicosapentaenoic acid in the patients' neutrophil cellular lipids decreased from 81:1 to 2.7:1, and the mean generation of leukotriene B4 (with calcium ionophore stimulation) significantly declined by 33%. The mean neutrophil chemotaxis to both leukotriene B4 and FMLP significantly increased toward the normal range at week 6. The generation of 5-lipoxygenase products by calcium ionophore-stimulated monocytes was not significantly suppressed, but a significant decline (37%) in platelet-activating factor generation was noted at week 6. The modulation of these measures of leukocyte inflammatory potential suggests that fish oil supplementation may have an antiinflammatory effect.

Effect of dietary enrichment with eicosapentaenoic and docosahexaenoic acids on in vitro neutrophil and monocyte leukotriene generation and neutrophil function.

The effects of dietary fish-oil fatty acids on the function of the 5-lipoxygenase pathway of peripheral-blood polymorphonuclear leukocytes and monocytes were determined in seven normal subjects who supplemented their usual diet for six weeks with daily doses of triglycerides containing 3.2 g of eicosapentaenoic acid and 2.2 g of docosahexaenoic acid. The diet increased the eicosapentaenoic acid content in neutrophils and monocytes more than sevenfold, without changing the quantities of arachidonic acid and docosahexaenoic acid. When the neutrophils were activated, the release of [3H]arachidonic acid and its labeled metabolites was reduced by a mean of 37 per cent, and the maximum generation of three products of the 5-lipoxygenase pathway was reduced by more than 48 per cent. The ionophore-induced release of [3H]arachidonic acid and its labeled metabolites from monocytes in monolayers was reduced by a mean of 39 per cent, and the generation of leukotriene B4 by 58 per cent. The adherence of neutrophils to bovine endothelial-cell monolayers pretreated with leukotriene B4 was inhibited completely, and their average chemotactic response to leukotriene B4 was inhibited by 70 per cent, as compared with values determined before the diet was begun and six weeks after its discontinuation. We conclude that diets enriched with fish-oil-derived fatty acids may have antiinflammatory effects by inhibiting the 5-lipoxygenase pathway in neutrophils and monocytes and inhibiting the leukotriene B4-mediated functions of neutrophils.