Treatment of multiple sclerosis with lofepramine, L-phenylalanine and vitamin B(12): mechanism of action and clinical importance: roles of the locus coeruleus and central noradrenergic systems.

In a randomized, placebo-controlled double-blind trial a combination of lofepramine, phenylalanine and vitamin B(12) was found to be effective in relieving the symptoms of multiple sclerosis (MS). The effect occurred within 2-4 weeks, and improved all types of symptoms in all types of MS. The combination was also effective in relieving symptoms in patients with chronic pain and chronic fatigue. We hypothesize that the action of this combined therapy may relate to activation of the noradrenergic locus coeruleus/lateral tegmentum (LC/LT) system which has the potential to influence the functioning of large areas of the brain and spinal cord.

Depression and bipolar disorder: relationships to impaired fatty acid and phospholipid metabolism and to diabetes, cardiovascular disease, immunological abnormalities, cancer, ageing and osteoporosis. Possible candidate genes.

Depression and bipolar disorder are two of the commonest illnesses in the developed world. While some patients can be treated effectively with available drugs, many do not respond, especially in the depression related to bipolar disorder. Depression is associated with diabetes, cardiovascular disease, immunological abnormalities, multiple sclerosis, cancer, osteoporosis and ageing: in each case depressed individuals have a worse outcome than non-depressed individuals. In all of these conditions there is now evidence of impaired phospholipid metabolism and impaired fatty acid-related signal transduction processes. Impaired fatty acid and phospholipid metabolism may be a primary cause of depression in many patients and may explain the interactions with other diseases. Several novel gene candidates for involvement in depression and bipolar disorder are proposed.

Concentration- and time-dependent effects of gamma-linolenic acid supplementation to tumor cells in culture.

Gamma-linolenic acid (GLA) supplemented to neuroblastoma SK-N-BE, tubal carcinoma TG and colon carcinoma SW-620 cells was incorporated into phospholipids in all the cell lines (although to different extents), in a concentration- and time-dependent manner. All the cell lines were able to metabolize GLA to arachidonic acid, SK-N-BE being the most active. Supplementation with low GLA concentrations for short periods was not sufficient to impair cell proliferation; only higher amounts of GLA had an anti-proliferative effect also in short times. In these conditions, the antiproliferative effect of GLA is probably due to cellular dysfunction caused by fatty acid modifications.

Effects of oral supplementation with evening primrose oil for six weeks on plasma essential fatty acids and uremic skin symptoms in hemodialysis patients.

Abnormalities in plasma composition of essential fatty acids (EFAs) may be associated with the etiology of pruritus and other skin problems in patients undergoing hemodialysis. To study whether an oral supplementation with omega-6 (n-6) EFAs would restore deranged plasma EFAs and ameliorate skin symptoms, 9 and 7 dialysis patients were randomly assigned to receive either gamma-linolenic acid (GLA)-rich evening primrose oil (EPO) or linoleic acid (LA) (2 g/day each) for 6 weeks. Plasma concentrations of EFA were analyzed by gas chromatography and uremic skin symptoms were assessed for dryness, pruritus and erythema by questionnaire and visual inspection in a double-blind manner. The patients given EPO exhibited a significant (p < 0.05) increase in plasma dihomo-gamma-linolenic acid (a precursor of anti-inflammatory prostaglandin E1) with no concomitant change in plasma arachidonic acid (a precursor of pro-inflammatory prostaglandin E2 and leukotriene B4). In contrast, those given LA exhibited a significant (p < 0.05) increase in LA but not in any other n-6 EFAs, whereas they exhibited a significant (p < 0.05) decrease in plasma docosahexaenoic acid. The patients given EPO showed a significant (p < 0.05) improvement in the skin scores for the three different uremic skin symptoms over the baseline values and a trend toward a greater improvement (0.05 < p < 0.1) in pruritus scores than those given LA. Results indicate that GLA-rich EPO would be a more favorable supplemental source than LA in terms of shifting eicosanoid metabolism toward a less inflammation status through modifying plasma concentrations of their precursor n-6 EFAs. Further studies are required to confirm the efficacy and safety of EPO therapy for the treatment of uremic pruritus.

Age-related changes in LTP and antioxidant defenses are reversed by an alpha-lipoic acid-enriched diet.

Among the age-related changes identified in rat hippocampus are impairments in LTP and glutamate release. These deficits have been coupled with decreased arachidonic acid concentration. In this study we compared LTP and glutamate release in groups of aged and young rats fed for 8 weeks on a control diet or on a diet enriched in alpha-lipoic acid. Dietary supplementation in aged rats restored hippocampal arachidonic acid concentration to levels observed in tissue prepared from young rats. We observed that aged rats that received the experimental diet sustained LTP in perforant path-granule cell synapses in a manner indistinguishable from young rats whereas the age-related impairment in glutamate release was reversed in synaptosomes prepared from dentate gyrus obtained from these rats. The evidence presented supports the hypothesis that the alpha-lipoic acid-enriched diet has antioxidant properties, because the age-related increase in superoxide dismutase activity and decrease in alpha-tocopherol concentration were reversed. The finding that the age-related increase in interleukin-1 (IL-1)beta concentration was also reversed suggests a possible role for this cytokine in ageing.

gamma-Linolenic acid blocks cell cycle progression by regulating phosphorylation of p27kip1 and p57kip2 and their interactions with other cycle regulators in cancer cells.

gamma-Linolenic acid (gamma-LA), a n-6 essential fatty acid, has been previously shown to affect cell cycle and growth of cancer cells. This study examined the effects of gamma-LA on the cell cycle and cycle regulators in human colon cancer HT115 and breast cancer MCF7 cells. Brief treatment of cancer cells (<2 h) with gamma-LA resulted in a decrease in the phosphorylation of both cell cycle inhibitors, p27kip1 and p57kip2 as shown by immunoprecipitation and Western blotting. Protein levels of both inhibitors were increased following a prolonged culture of cells with the fatty acid. A co-precipitation study showed that in cells treated with gamma-LA there was an increase in the binding of these inhibitors with CDK4, CDC2, and cyclin E. Flow cytometry study indicated an inhibition of cell cycle progression by gamma-LA (G0/G1 -45.4%, S - 34.6%, G2+M - 20.0% in control, and 70.5%, 21.0%, and 8.5%, respectively, in gamma-LA treated cells). It is concluded that gamma-linolenic acid inhibits cell cycle progression in the cancer cell lines investigated, via its regulation of the phosphorylation and subsequent degradation of p27kip1 and p57kip2 and their interactions with other cycle regulators.

The effects of n-6 polyunsaturated fatty acids on the expression of nm-23 in human cancer cells.

This study examined the effect of n-6 polyunsaturated fatty acids (PUFAs) on the expression of nm-23, a metastasis-suppressor gene, in two highly invasive human cancer cell lines, HT115 and MDA MB 231. A range of n-6 and n-3 PUFAs were tested. We report that while linoleic acid and arachidonic acid reduced the expression of nm-23-H1, gamma linolenic acid (GLA) and its soluble lithium salt markedly increased the expression of the molecules. The stimulation of the expression of nm-23 by GLA was seen at both protein and mRNA levels. Up-regulation of nm-23 was also associated with a reduction of the in vitro invasiveness of these cells. It is concluded that gamma linolenic acid (GLA) enhances the expression of nm-23. This contributes to the inhibition of the in vitro invasion of tumour cells.

Regulation of tight junction permeability and occludin expression by polyunsaturated fatty acids.

Tight junctions (TJ) are the topical most structure in epithelial and endothelial cells and play a key role in the control of permeability and prevention of tumour cell invasion of endothelium. In this study we examined the effects of a range of polyunsaturated fatty acids on the function of TJs and the expression of occludin, a key molecule in the TJs of the human vascular endothelial cell line, ECV304. Treatment of the endothelial cells with gamma linolenic acid, an anti-cancer PUFA, increased the transendothelial cell resistance (TER) and reduced the paracellular permeability to large molecules. The effects were seen without any changes in the viability of the endothelial cells. Occludin, a recently identified molecule, which plays a major role in tight junctions was up-regulated by this fatty acid as revealed by both Western blotting and immunofluorescence. Other fatty acids were also tested. Eicosapentaenoic acid (EPA) also exerted an up-regulatory effect, but LA and AA down-regulated the expression. We conclude that GLA and EPA which also have other anti-cancer effects, regulate the expression of occludin in endothelial cells and thus contribute to the modification of the TER of these cells.

The effects of lithium gamma-linolenic acid in reversing LPBM5 MuLV induced suppression of hematopoietic progenitor cells in vitro.

Lithium gamma linolenic acid (Li-GLA), was evaluated for its possible role as an antiviral agent. Li-GLA 15 micrograms ml-1 was administered to both normal and LP-BM5 MuLV retroviral infected murine bone marrow cultures. After 2 weeks of treatment, numbers of progenitors being produced by infected/treated cultures were reduced to some 10% that of normal cultures. In the remaining 4 weeks, numbers of CFU-GM and BFU-E hematopoietic progenitors returned within normal range. The efficacy of Li-GLA in relieving retroviral hematopoietic bone marrow suppression correlates to a reduction in interleukin-4 (IL-4) secretion, normally elevated in association with LP-BMP5 infection. These data indicate that this reduction in bone marrow suppression of LP-BMP5 infected cells may be due to a killing of infected cells by the Li-GLA, rather than stimulating hematopoiesis as with other lithium compounds. To conclude this may indicate the possible dual effect of administration of LiGLA to virally infected individuals in reducing viral titre and to lower the toxicities associated with long term drug therapy.

Manipulation of lipid composition of rat heart myocytes aged in culture and its effect on alpha1-adrenoceptor stimulation.

The fatty acid composition of the phosphoinositides was evaluated in cultured neonatal rat cardiomyocytes during the aging-like process in vitro, comparing data obtained from control and gamma-linolenic acid supplemented cardiomyocytes. The response to alpha1 stimulation was evaluated in both control and supplemented cells to verify the relationship between the alterations of the phosphoinositide fatty acid composition concomitant to culture aging and the cell response to exogenous stimuli. Arachidonate level decreased as a function of age in all the phosphoinositides, which appeared to be more saturated as cells aged in culture. Inositol phosphate production in response to alpha1 stimulation decreased as cells aged in culture. Supplementation of culture medium with gamma-linolenic acid caused significant modifications in the fatty acid pattern of the phosphoinositides, which appeared less saturated than the corresponding fractions isolated from unsupplemented cells during the aging-like process. The modifications induced by the supplementation in the phosphoinositide fatty acid composition prevented the age-related reduction of inositol phosphate production upon stimulation. These results clearly indicate a major role for the lipid composition in determining the response to alpha1 stimulation, suggesting a nutritional approach to overcome some of the impairments of molecular events related to the process of aging.

Regulation of desmosomal cell adhesion in human tumour cells by polyunsaturated fatty acids.

Desmosomes are key structures in cell-cell adhesion. In this study we examined the effect of n-6 essential fatty acids on the expression of desmoglein (Dsg), desmosomal cadherin and the formation of desmosomes in E-cadherin negative human breast, colon and lung cancer cells and melanoma cells. Electron microscopy revealed that cells cultured with gamma linolenic acid (GLA) showed increased cell-cell adhesion together with an increase in the formation of desmoglein-containing desmosomes. Western blotting studies of cellular proteins demonstrated that, following culture with fatty acids, Dsg expression was modified, with the greatest increase seen after GLA treatment. Other fatty acids increased Dsg expression, but to a lesser extent. It is concluded that GLA regulates desmosome-mediated cell-cell adhesion in human cancer cells, particularly in cells without E-cadherin.

Gamma linolenic acid regulates expression of maspin and the motility of cancer cells.

Maspin, mammary serine protease inhibitor, is a recently identified tumour suppressor and has a profound effect on cell motility. This study examined the effect of gamma linolenic acid (GLA), an essential fatty acid (EFA) with anticancer properties, on the expression of maspin and motility of cancer cells. Six human cell lines including colon cancer, mammary cancer, and melanoma were used. Expression of maspin protein was determined by immunocytochemistry & Western blotting. Maspin mRNA was detected with reverse transcription-PCR (RT-PCR). Four of the six cell types expressed maspin with MDA MB 231 and ECV304 (endothelial cell) being negative. Treatment of these maspin positive cells with gamma linolenic acid (GLA) resulted in a concentration dependent stimulation of the expression of maspin protein with the effects seen as early as 4 hours. Linoleic acid had an inhibitory effects. Alpha linolenic acid and arachidonic acid had no significant effect. The mRNA levels from cells treated with GLA was seen to increase as shown by RT-PCR. Cell motility, monitored with time-lapse video recording and Hoffmann microscopy, showed a marked reduction in terms of spreading and migration on extracellular matrix coated surface. This reduction was reversed with anti-maspin antibody. It is concluded that GLA, a member of then-6 series of EFAs, up-regulates the expression of maspin which is associated with a reduction in the motility of cancer cells.

The importance of linoleic acid metabolites in cancer metastasis and in the synthesis and actions of 13-HODE.

Large scale human epidemiological studies indicate that high intakes of linoleic acid protect against the development of cancer. One mechanism may be the generation of 13-HODE from linoleic acid. 13-HODE prevents cell adhesion to endothelial cells and can inhibit cancer metastasis. 13-HODE synthesis is enhanced by cyclic AMP. Gamma-linolenic acid, a desaturated metabolite of linoleic acid, causes substantial stimulation of 13-HODE synthesis. A fall in gamma-linolenic acid synthesis with age may be related to the age-related fall in 13-HODE formation.

Mechanisms of the selective cytotoxic actions of certain essential fatty acids.

Polyunsaturated fatty acids (PUFA) have a selective cytotoxic/cytostatic effect on a number of tumor cell lines in culture. Although this process may be enhanced by the addition of iron there is a minimum level of PUFA necessary for potentiation of cell death. Vitamin E blocks PUFA cytotoxicity when added up to 5 days after fatty acid administration. Levels of thio-barbiturate reactive material (TBARM) in the medium rise in parallel with cell death. However, they are not affected by small alterations in temperature or oxygen tension. Incubating cells with PUFA causes marked alterations in the fatty acid patterns of both neutral and phospholipid fractions. Membrane fluidity is increased and the activity of membrane-bound receptors may be influenced directly or through the actions of eicosanoids derived from the exogenous fatty acid. PUFA may be an effective way of influencing tumor growth and a safe approach for the management of human cancer.

Calcium metabolism, osteoporosis and essential fatty acids: a review.

Essential fatty acid (EFA)-deficient animals develop severe osteoporosis coupled with increased renal and arterial calcification. This picture is similar to that seen in osteoporosis in the elderly, where the loss of bone calcium is associated with ectopic calcification of other tissues, particularly the arteries and the kidneys. Recent mortality studies indicate that the ectopic calcification may be considerably more dangerous than the osteoporosis itself, since the great majority of excess deaths in women with osteoporosis are vascular and unrelated to fractures or other bone abnormalities. EFAs have now been shown to increase calcium absorption from the gut, in part by enhancing the effects of vitamin D, to reduce urinary excretion of calcium, to increase calcium deposition in bone and improve bone strength and to enhance the synthesis of bone collagen. These desirable actions are associated with reduced ectopic calcification. The interaction between EFA and calcium metabolism deserves further investigation since it may offer novel approaches to osteoporosis and also to the ectopic calcification associated with osteoporosis which seems to be responsible for so many deaths.

gamma-Linolenic acid supplementation can affect cancer cell proliferation via modification of fatty acid composition.

We examined the effect of gamma-linolenic acid (GLA) supplementation on the growth and fatty acid composition of three human tumor cell lines (the neuroblastoma CHP-212, the tubal carcinoma TG, and the colon carcinoma SW-620), in order to evaluate the relationship between GLA-induced tumor cell death and the distribution of fatty acids in tumor cells. At the highest GLA concentrations (10 and 20 micrograms/ml), the DNA synthesis was completely abolished; at 5 micrograms/ml GLA only SW-620 cells did not proliferate, while CHP-212 and TG cells showed a residual [3H]-thymidine incorporation. GLA levels were very low in cells grown in control medium; GLA supplementation caused a significant incorporation of GLA itself in all the cell lines at each concentration. In TG and CHP-212 cells, GLA was metabolized, although to a different extent, to dihomo-gamma linolenic acid and arachidonic acid. SW-620 cells neither elongated nor desaturated the incorporated GLA. The highest cytostatic effect was reached when GLA was not transformed into its metabolites, suggesting that the GLA toxicity to tumor cells is not dependent on metabolites but is due to GLA itself.

Essential fatty acids and abnormal involuntary movements in the general male population: a study of men born in 1933.

Spontaneous and tardive dyskinesias were studied in a random population sample of 446 men, aged 59 years. Dyskinesia, defined as an AIMS score of at least 2 in any body part, was seen in 15.1% (n = 74). Dyskinetic men had a higher cigarette consumption, and they had higher frequencies of psychiatric morbidity and exposure to neuroleptics. Dyskinesia was also associated with several abnormalities in EFA concentrations in plasma, but the most consistent finding was the low arachidonic acid levels in phospholipids, triglycerides and cholesterol esters. In a logistic regression model, cigarette consumption (P < 0.02), exposure to neuroleptics (P < 0.01), and low arachidonic acid levels in the phospholipid fraction (P < 0.0001) were independently associated with dyskinesia.

Inhibition of neutrophil respiratory burst and cytokine priming by gamma-linolenic acid.

The effect of n-6 fatty acids, particularly gamma-linolenic acid (GLA), on the oxidase response and neutrophil priming by tumour necrosis factor alpha and interleukin 8 was studied in both normal volunteers and patients with obstructive jaundice. GLA inhibited the neutrophil respiratory burst at concentrations higher than 50 mummol/l, but abolished cytokine priming at concentrations as low as 1 mummol/l. Inhibition was not the result of either cytotoxicity to the neutrophils or alteration in cytosolic free calcium homoeostasis. It is concluded that GLA is a potential inhibitor of neutrophil priming by cytokines and of the oxidative response.