The development of an ELISA for group IVA phospholipase A2 in human red blood cells.

An immunoassay for IVA phospholipase A2 in human red blood cells is described. The assay is a non-competitive sandwich assay in which increasing amounts of the measured protein produce increased luminescence. The antibodies used in the assay are directed against two unique epitopes of the molecule, which sequentially trap and detect the protein. The standard curve covers the range 0.7ng to 23ng/mL (0.07 to 2.3ng/well). The intra-assay and inter-assay coefficients of variation were 9% and 12%, respectively. Evidence is presented that the assay is specific for the alpha paralog of IV PLA2. The assay allows simple and rapid quantification of IVAPLA2 in red blood cell lysates and other biological fluids.

Long-chain polyunsaturated fatty acids modulate interleukin-1beta-induced changes in behavior, monoaminergic neurotransmitters, and brain inflammation in rats.

Recent evidence has suggested that an imbalance between membrane (n-3) and (n-6) fatty acids may contribute to the etiology of autoimmune and neurodegenerative diseases. In this study, the mechanisms by which eicosapentaenoic acid (EPA), gamma-linolenic acid (GLA), and arachidonic acid (AA) modulate neurotransmitters, behavior, and brain inflammation were evaluated in rats that received central saline or interleukin-1beta (IL-1) administrations. In rats treated with saline, only the AA-enriched diet significantly increased anxiety-like behavior in the elevated plus maze, which was associated with increased corticosterone secretion. AA also increased the turnover of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) in the amygdala and increased the prostaglandin (PG)E(2) level in the hippocampus. IL-1 administration slowed rat learning in the water maze and increased anxiety-like behavior, changes which were associated with increased homovanillic acid and 5-HT turnover, decreased NA in the hippocampus and amygdala, decreased DA in the frontal cortex, and decreased IL-10 in limbic brain regions. Increased corticosterone secretion following IL-1 administration was accompanied by increased NA turnover in the hippocampus (P < 0.05) and increased PGE(2) concentration (P < 0.01) in the limbic brain regions. Of the 3 diets tested, only EPA attenuated IL-1-induced behavioral changes (P < 0.05 or 0.01), which was associated with the modulation of EPA on the neuroendocrine and immune changes induced by IL-1. GLA reduced hippocampal PGE(2) concentration in rats given IL-1 (P < 0.01). AA did not counteract any of the changes induced by IL-1. These results suggest that EPA, GLA, and AA play different roles in the neuroendocrine-immune network.

Neuroprotective actions of eicosapentaenoic acid on lipopolysaccharide-induced dysfunction in rat hippocampus.

Eicosapentaenoic acid (EPA) protects hippocampus from age-related and irradiation-induced changes that lead to impairment in synaptic function; the evidence suggests that this is due to its anti-inflammatory effects, specifically preventing changes induced by the proinflammatory cytokine, interleukin-1beta (IL-1beta). In this study, we have investigated the possibility that EPA may prevent the effects of lipopolysaccharide (LPS) administration, which have been shown to lead to deterioration of synaptic function in rat hippocampus. The data indicate that treatment of hippocampal neurones with EPA abrogated the LPS-induced increases in phosphorylation of the mitogen-activated protein kinase, c-Jun N-terminal kinase (JNK), the transcription factor, c-Jun and the mitochondrial protein, Bcl-2. In parallel, we report that intraperitoneal administration of LPS to adult rats increases phosphorylation of JNK, c-Jun and Bcl-2 in hippocampal tissue and that these changes are coupled with increased IL-1beta concentration. Treatment of rats with EPA abrogates these effects and also blocks the LPS-induced impairment in long-term potentiation in perforant path-granule cell synapses that accompanies these changes. We propose that the neuroprotective effect of EPA may be dependent on its ability to inhibit the downstream consequences of JNK activation.

Ethyl-EPA in Alzheimer's disease--a pilot study.

This pilot study of ethyl-eicosapentaenoate (ethyl-EPA) in the treatment of Alzheimer's disease aimed to estimate the magnitude of any change in measures of cognition during a 12-week treatment period. A simple linear design was used in which each patient had a baseline period of 12 weeks without treatment, followed by 12 weeks' treatment with ethyl-EPA. Blood samples were taken both before and after the treatment period to measure erythrocyte membrane fatty acids. Assessments comprised cognitive measures and visual analogue ratings of overall assessment of functioning. There was little difference between treatment and baseline periods in the rate of decline of efficacy measures, except for a small improvement in carer's visual analogue rating (P=0.02). It was concluded that it is unlikely there were any clinically important treatment effects of ethyl-EPA on cognition during the 12-week treatment period. A longer treatment period may be necessary to demonstrate efficacy of ethyl-EPA in this disorder.

A pilot open case series of ethyl-EPA supplementation in the treatment of anorexia nervosa.

Anorexia nervosa (AN) carries the highest risk of morbidity and mortality amongst psychiatric disorders. The efficacy of current treatment approaches is limited. Despite the fat-phobic nature of the disease, poly-unsaturated fatty acids (PUFAs) have not received much research attention. Patients who consume western diet, which is rich in n-6 PUFAs and trans-fatty acids, are likely to develop severe n-3 PUFA deficiency during self-induced starvation. Re-feeding programmes do not take into consideration n-3 EFA intake, possibly leading to further n-3 PUFA deficiency during weight restoration, and this might contribute to the maintenance of the disorder. To test this hypothesis, we carried out a systematic case series of E-EPA supplementation in the treatment of AN. Seven young patients received 1g/day E-EPA in addition to standard treatment, and were followed up for 3 months. Three of them recovered and four improved. Randomised controlled trials are warranted to examine the effectiveness of E-EPA in AN further.

Dietary ethyl-eicosapentaenoic acid but not soybean oil reverses central interleukin-1-induced changes in behavior, corticosterone and immune response in rats.

Omega (n)-3 and n-6 fatty acids are important membrane components of neurons and immune cells, and related to psychiatric and inflammatory diseases. Increased ratio of n-6/n-3 in the blood has been reported in depressed patients and in students following stress exposure. The n-3 fatty acid, eicosapentaenoic acid (ethyl-EPA) suppresses inflammation and has antidepressant properties. Interleukin (IL)-1beta can stimulate corticosterone secretion, induce anxiety and stress-like behavior and inflammatory responses. This study was to evaluate the effect of diets enriched with coconut oil, ethyl-EPA and soybean oil on central IL-1beta induced stress and anxiety-like behavior, induced changes in the concentration of prostaglandin (PG) E2 and corticosterone and the release of IL-10. Groups of rats were fed with either 5% coconut oil (as control diet), 0.2% EPA with 4.8% coconut oil or 1% EPA with 4% coconut oil and 5% soybean oil for 7 weeks. The central administration of IL-1beta induced sickness, stress and anxiety-like behavior as indicated by a reduction in body weight, decreased time spent, and the number of entries, into the open arms of the elevated plus maze and decreased exploration and entry into the central zone of the "open field" apparatus. IL-1beta also increased PGE2 and corticosterone concentrations and decreased the release of IL-10 from leucocytes. Food enriched with ethyl-EPA but not soybean oil, significantly attenuated most of these changes. These results demonstrate that ethyl-EPA has anti-inflammatory, anti-stress and anti-anxiety effects in rats.

Ethyl-eicosapentaenoate and dexamethasone resistance in therapy-refractory depression.

Preliminary evidence shows that ethyl-eicosapentaenoate (E-EPA) has a marked clinical effect when used as an adjunct in therapy-refractory depression. EPA belongs to the class of polyunsaturated omega-3 fatty acids. The mechanism of its action in depression is not fully understood. There are two related fields where the pathophysiology of refractory depression meets the effect of EPA. First, a general immunosuppressive effect of EPA meets a general immunoactivation in severe depression, especially an increase in CD4/CD8 ratio, neutrophilia, and an increase in interleukins (IL)-6 and IL-12 and of prostaglandin E2 (PGE2). Secondly, a resistance to dexamethasone (Dex) suppression of the HPA axis meets the effects of EPA on multidrug resistance reversing and HPA axis suppression. The effects of EPA on the immune system, the HPA axis, and multidrug resistance are connected through the action of a transport protein called p-glycoprotein (p-gp). Physiological and synthetic steroids such as cortisol and Dex are substrates of p-gp, and so Dex resistance in depression may be related to dysfunction of this protein. In addition, expression of p-gp is induced by PGE2, and EPA inhibits the synthesis of PGE2. The reversal of drug resistance by EPA may be mediated via this immunological mechanism and lead to its antidepressive efficacy. In addition, antidepressants such as amitriptyline, which have special efficacy in severe depression, decrease p-gp function. EPA may, furthermore, enhance the action of antidepressants, like many SSRIs that are p-gp substrates, which are actively transported out of the intracerebral space at the level of the blood-brain barrier.

Cytosolic phospholipase A2 type IVA is present in human red cells.

Phospholipase A(2) type IVA (IVAPLA(2)) is a cytosolic enzyme that on activation selectively releases arachidonic acid (AA) from cell membrane phospholipids. Both AA and lysophospholipid, products of the enzymic reaction, can function as signal transducers in cellular interactions. The enzyme is present in most cells, including polymorphs, eosinophils, and platelets. This study used affinity purification to extract IVAPLA(2) from red cell lysate prepared from leukocyte- and platelet-depleted human blood to overcome the masking effect of hemoglobin on Western blot detection. We show that IVAPLA(2) is present in red cells as a 90-kDa protein.

Effects of dietary n-3 or n-6 fatty acids on interleukin-1beta-induced anxiety, stress, and inflammatory responses in rats.

The present study demonstrated that an omega (n)-3 fatty acid, ethyl-eicosapentaenoic acid (ethyl-EPA), supplemented diet significantly attenuated the stress/anxiety behavior of rats in the "open field" and elevated plus maze, which was induced by subchronic intracerebroventricular administration of proinflammatory cytokine interleukin (IL)-1beta. Ethyl-EPA also reduced the rise in serum corticosterone induced by IL-1. The n-6 fatty acid ethyl-gamma-linolenic acid (ethyl-GLA) had little effect on the IL-1-induced changes in behavior and the corticosterone concentration. Following IL-1beta administration, ethyl-EPA reduced the elevated prostaglandin (PG) E2 secretion and increased the secretion of antiinflammatory cytokine IL-10 from whole blood cells. Ethyl-GLA showed a similar antiinflammatory effect to ethyl-EPA. By contrast, n-6 fatty acid arachidonic acid (AA) had no effect on the behavior, immune, and endocrine changes induced by IL-1. AA alone enhanced the basal inflammatory response, raised serum corticosterone concentrations, and induced anxiety behavior in the elevated plus maze. The reduced growth rates of rats following the administration of IL-1 was attenuated by ethyl-EPA, and to a greater extent by ethyl-EPA plus ethyl-GLA, but not by AA alone or in combination with ethyl-EPA. Thus, ethyl-EPA would appear to antagonise the endocrine, immune, and behavioral effects of subchronic IL-1 administration. Ethyl-GLA only antagonised IL-1-induced inflammatory changes, whereas AA caused an increase in the secretion of corticosterone and PGE2, and induced anxiety-like behavior without enhancing the effects of IL-1.

A low toxicity maintenance regime, using eicosapentaenoic acid and readily available drugs, for mantle cell lymphoma and other malignancies with excess cyclin D1 levels.

Mantle cell lymphoma is a difficult to treat non-Hodgkin's lymphoma (NHL) whose biochemistry is unusually well characterised. Almost all and perhaps all patients overexpress the cyclin D1 protein which is crucial in driving cells from the G1 to the S phase. This overexpression may be responsible for the refractoriness. Despite this understanding, treatments for mantle cell lymphoma are based on standard NHL regimes of cyclophosphamide, doxorubicin, vincristine and prednisone, perhaps supplemented with the monoclonal antibody rituximab. There has never been any attempt to direct treatment to the cyclin D1 mechanism or to angiogenesis which is now known to be important in all lymphomas. Both these targets lend themselves to long-term maintenance regimes of relatively low toxicity which can be used as adjuvants to standard therapy. Agents which have recently been shown to block cyclin D1 translation by regulating calcium levels are the unsaturated essential fatty acid, eicosapentaenoic acid (EPA), the antidiabetic thiazolidinediones, and the antifungal agent, clotrimazole. Two types of agent which have been shown to inhibit angiogenesis are the teratogen, thalidomide, and the selective inhibitors of cyclo-oxygenase 2 (COX-2). Retinoids exert synergistic effects with EPA and have been shown to inhibit both tumour growth and angiogenesis. The mechanisms of action of these various agents are discussed, and specific suggestions are made for low toxicity maintenance therapy of mantle cell lymphoma and of other tumours which overexpress cyclin D1.

Essential polyunsaturated fatty acid and lipid peroxide levels in never-medicated and medicated schizophrenia patients.

BACKGROUND: Reduced levels of membrane essential polyunsaturated fatty acids (EPUFAs) and increased levels of lipid peroxidation products (thiobarbituric acid reactive substances; TBARS) have been observed in chronic medicated schizophrenics. The relationship of EPUFA and TBARS to psychopathology is unclear, since their levels may be altered differentially by duration of illness and antipsychotic treatment. To minimize these confounds, their levels were compared among never-medicated patients in early illness, medicated patients and control subjects with similar lifestyle and common ethnic background. METHODS: RBC membrane EPUFAs, plasma TBARS, and various dimensions of psychopathology were measured using established procedures in never-medicated (n = 20) and medicated (n= 32) schizophrenia patients and in control subjects (n= 45). RESULTS: Reduced levels of EPUFAs, particularly arachidonic acid (AA) and docosahexaenoic acid (DHA), were found in never-medicated compared with control subjects; however, the reductions in levels of both AA and DHA were much smaller in medicated versus never-medicated patients; AA levels were similar to levels in control subjects. Only DHA levels were significantly reduced in medicated patients. Lower membrane AA levels were associated with increased levels of plasma TBARS in never-medicated patients. Lower levels of membrane EPUFAs and higher levels of plasma TBARS were associated with the severe symptoms in never-medicated versus medicated patients. CONCLUSIONS: Data indicate that reduced EPUFAs and increased TBARS exist in never-medicated patients, and these measures correlate with the severity of psychopathology indicating that the membrane EPUFA status may reflect the outcome of schizophrenia.

A new category of psychotropic drugs: neuroactive lipids as exemplified by ethyl eicosapentaenoate (E-E).

New treatments for psychiatric disorders are urgently required. Recent reviews show that there have been no improvements in efficacy of drugs for either affective disorders or schizophrenia since the first compounds were introduced over 40 years ago. Neuroactive lipids represent an entirely novel class of psychotropic compounds. Ethyl eicosapentaenoate is the first example of this group. Placebo-controlled studies have found it to be effective in depression, in treatment-unresponsive schizophrenia and in tardive dyskinesia. It is extremely well tolerated with none of the usual side-effects of either antidepressants or neuroleptics. It probably works by modulating postreceptor signal transduction processes.

A dose-ranging study of the effects of ethyl-eicosapentaenoate in patients with ongoing depression despite apparently adequate treatment with standard drugs.

BACKGROUND: In depressed patients, low blood levels of eicosapentaenoic acid are seen. We tested the antidepressive effect of ethyl-eicosapentaenoate in these patients. METHODS: We included 70 patients with persistant depression despite ongoing treatment with an adequate dose of a standard antidepressant. Patients were randomized on a double-blind basis to placebo or ethyl-eicosapentaenoate at dosages of 1, 2, or 4 g/d for 12 weeks in addition to unchanged background medication. Patients underwent assessment using the 17-item Hamilton Depression Rating Scale, the Montgomery-Asberg Depression Rating Scale, and the Beck Depression Inventory. RESULTS: Forty-six (88%) of 52 patients receiving ethyl-eicosapentaenoate and 14 (78%) of 18 patients receiving placebo completed the 12-week study with no serious adverse events. The 1-g/d group showed a significantly better outcome than the placebo group on all 3 rating scales. In the intention-to-treat group, 5 (29%) of 17 patients receiving placebo and 9 (53%) of 17 patients receiving 1 g/d of ethyl-eicosapentaenoate achieved a 50% reduction on the Hamilton Depression Rating Scale score. In the per-protocol group, the corresponding figures were 3 (25%) of 12 patients for placebo and 9 (69%) of 13 patients for the 1-g/d group. The 2-g/d group showed little evidence of efficacy, whereas the 4-g/d group showed nonsignificant trends toward improvement. All of the individual items on all 3 rating scales improved with the 1-g/d dosage of ethyl-eicosapentaenoate vs placebo, with strong beneficial effects on items rating depression, anxiety, sleep, lassitude, libido, and suicidality. CONCLUSION: Treatment with ethyl-eicosapentaenoate at a dosage of 1 g/d was effective in treating depression in patients who remained depressed despite adequate standard therapy.