Omega-3 fatty acids and neurological injury

Studies with omega-3 polyunsaturated fatty acids (PUFA) have shown that these compounds have therapeutic potential in several indications in neurology and psychiatry. Acute spinal cord injury (SCI) is an event with devastating consequences, and no satisfactory treatment is available at present. The pathogenetic mechanisms associated with SCI include excitotoxicity, increased oxidation and inflammation. We review here our recent studies, which suggest that omega-3 PUFA have significant neuroprotective potential in spinal cord trauma. In a first study, we administered an intravenous bolus of alpha-linolenic acid (LNA) or docosahexaenoic acid (DHA) 30 min after spinal cord hemisection injury in adult rats. The omega-3 PUFA led to increased neuronal and glial survival, and a significantly improved neurological outcome. In subsequent studies, we tested DHA in a more severe compression model of SCI. We also explored a combined acute and chronic treatment regime using DHA. Saline or DHA was administered intravenously 30 min after compression of the spinal cord. After injury, the saline group received a standard control diet, whereas DHA-injected animals received either a control or a DHA-enriched diet for 6 weeks following injury. We assessed locomotor recovery and analysed markers for cell survival and axonal damage, and we also investigated the effects of the treatment on the inflammatory reaction and the oxidative stress that follow SCI. We showed that the acute DHA treatment is neuroprotective after compression SCI, even if the treatment is delayed up to an hour after injury. The DHA injection led to an increased neuronal and glial cell survival, and the effect of the DHA injection was amplified by addition of DHA to the diet. Rats treated with a DHA injection and a DHA-enriched diet performed significantly better at 6 weeks in terms of neurological outcome. The analysis of the tissue after DHA administration showed that the fatty acid significantly reduced lipid peroxidation, protein oxidation and RNA/DNA oxidation, and the induction of COX-2. Parallel studies in a facial nerve injury model in mice also showed pro-regenerative effects of chronic dietary administration of DHA after nerve lesion. These observations suggest that treatment with omega-3 PUFA could represent a promising therapeutic approach in the management of neurological injury.     

The ketogenic diet increases mitochondrial glutathione levels

The ketogenic diet (KD) is a high-fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague–Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of β-hydroxybutyrate (BHB). KD-fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet-fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate-limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD-fed rats, as well as up-regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD-fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H₂O₂ production and H₂O₂-induced mtDNA damage. Isolated hippocampal mitochondria from KD-fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H₂O₂ production and mtDNA damage. Together, the results demonstrate that the KD up-regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant-induced damage.     

Omega-3 fatty acids and neuropsychiatric disorders

Epidemiological evidence suggests that dietary consumption of the long chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), commonly found in fish or fish oil, may modify the risk for certain neuropsychiatric disorders. As evidence, decreased blood levels of omega-3 fatty acids have been associated with several neuropsychiatric conditions, including Attention Deficit (Hyperactivity) Disorder, Alzheimer's Disease, Schizophrenia and Depression. Supplementation studies, using individual or combination omega-3 fatty acids, suggest the possibility for decreased symptoms associated with some of these conditions. Thus far, however, the benefits of supplementation, in terms of decreasing disease risk and/or aiding in symptom management, are not clear and more research is needed. The reasons for blood fatty acid alterations in these disorders are not known, nor are the potential mechanisms by which omega-3 fatty acids may function in normal neuronal activity and neuropsychiatric disease prevention and/or treatment. It is clear, however, that DHA is the predominant n-3 fatty acid found in the brain and that EPA plays an important role as an anti-inflammatory precursor. Both DHA and EPA can be linked with many aspects of neural function, including neurotransmission, membrane fluidity, ion channel and enzyme regulation and gene expression. This review summarizes the knowledge in terms of dietary omega-3 fatty acid intake and metabolism, as well as evidence pointing to potential mechanisms of omega-3 fatty acids in normal brain functioning, development of neuropsychiatric disorders and efficacy of omega-3 fatty acid supplementation in terms of symptom management.     

N-3 fatty acids in the Mediterranean diet

Fats in the diet of countries in the Mediterranean basin are typically represented by olive oil, but the high consumptions of vegetables and to some extent also of fish result in appreciable intakes of n-3 fatty acids. In fact, various plant foods are relatively rich in the 18 carbon n-3 fatty acid, alpha linolenic acid, ALA, while the generally moderate consumption of fish, except for certain communities living close to the sea, contributes to the intake of the long-chain n-3. Although the amounts of fats in ALA-containing plant foods are low, the relatively high concentrations of this fatty acid and the large size of the portions consumed allow to reach appreciable doses of ALA, an n-3 fatty acid that has been shown to exert favourable effects on various relevant factors in cardiovascular protection. In addition, consumption of relatively small amounts of certain typical dry fruit components of the diet such as walnuts, provides a sizable supply of ALA that is also rather efficiently converted to the ALA derivative eicosapentaenoic acid (EPA). Additional rather typical wild food components of the diet in certain countries, i.e. snails and frogs, are also appreciable sources of ALA. It appears thus that the consumption of typical Mediterranean foods provides relevant intakes of n-3 fatty acids, especially ALA, that appears to be efficiently absorbed and also transformed at least to the long-chain derivative EPA.     

游离脂肪酸受体蛋白研究进展

游离脂肪酸不仅是人和动物体的一种重要能量来源,也是一种重要的信号分子。最近研究表明,游离脂肪酸受体蛋白在维持机体内的葡萄糖稳衡、脂肪形成、白细胞功能等生理过程中都有重要的作用,对于调控人或动物的营养代谢及疾病发生具有重要生理意义。     

Ingensin, a fatty acid-activated serine proteinase from rat liver cytosol

The enzyme responsible for the succinylleucylleucylvalyltyrosine methylcoumarylamide- (SLLVT-) degrading activity was purified from the postmitochondrial supernatant of rat liver (Yamamoto, T., Nojima, M., Ishiura, S. and Sugita, H. (1986) Biochim. Biophys. Acta 882, 297-304). The enzyme, named ingensin, was activated by saturated fatty acids, especially myristic acid, as well as by unsaturated linoleic acid and arachidonic acid. Although 2-mercaptoethanol activated ingensin 2-fold and p-chloromercuribenzoate and HgCl2 completely inhibited its peptide-hydrolyzing activity, the enzyme is activated by the addition of a thiol-blocking reagent, monoiodoacetic acid. Ingensin was also inhibited by a specific serine proteinase inhibitor, diisopropyl fluorophosphate, but not by a specific cysteine proteinase inhibitor, E-64-c. These results suggest that the enzyme is a serine proteinase with an active thiol group(s) near the active site. We have found that the addition of glycerol and nordihydroguaiaretic acid lowered the extent of its activation by fatty acids as well as its intrinsic peptide-hydrolyzing activity.     

Modulation of human erythrocyte shape and fatty acids by diet

A semi-synthetic diet (Vivonex) was administered via nasogastric tube to three cystic fibrosis patients with pancreatic exocrine deficiency for 14 days to gain weight. Dietary essential fatty acids were provided as safflower oil, which constituted 1.3% of total calories. Plasma and red blood cells were analyzed for the content and composition of lipids at the start of the diet and at days 7 and 14 of the dietary period, and the results were correlated with the morphology of the cells. Feeding Vivonex to the patients led to an essential fatty acid deficiency, which was manifested in a 50% decrease in the linoleic acid content of the phosphatidylcholine of plasma and red blood cells at days 7 and 14 and in a 20% decrease in the linoleic acid content of red cell phosphatidylethanolamine at day 14. There was no significant alteration in the levels or composition of the other phospholipid classes and in the free cholesterol/phospholipid ratio. The decrease in the linoleic acid content of the erythrocytes was accompanied by a dramatic increase in the proportion of cells as echinocytes. We conclude that restricted linoleic acid availability in cystic fibrosis patients causes a change in red blood cell shape either directly by decreasing the linoleoylphosphatidylcholine content of the membrane or indirectly by affecting enzyme activity.     

The ketogenic diet changes metabolite levels in hippocampal extracellular fluid

Despite successful use of the ketogenic diet (KD) for the treatment of drug-resistant epilepsy, its mechanism of action is unclear. After KD-feeding, increased plasma D-beta-hydroxybutyrate (BHB) levels appear to be important for protection against seizures. We hypothesized that the KD leads to metabolic changes in the brain, which are reflected in the hippocampal extracellular fluid (hECF). CD1 mice were fed control or KD for 2-3 weeks since weaning. In vivo microdialysis of hECF was used to measure the levels of glucose, lactate, as well as BHB under basal conditions and during 30 min stimulation with 60 mM K(+), which was retrodialysed. The hECF BHB concentration in KD-fed mice was determined as 43.4±10.1 μM using the zero-flow method and 50.7±5.5 μM based on in vitro recovery. The total BHB concentration in brain homogenate from KD-fed mice was 180 nmol/g. The intracellular BHB concentration is therefore estimated to be about 3-fold higher than the extracellular level, which suggests that BHB in adolescent mouse brains may not be quickly metabolized. The basal hECF glucose concentration was 30% lower in KD-fed mice, indicating that glucose may be less important as an energy source. Lactate levels were similar in control and KD-fed mice. High potassium stimulation elevated lactate by 3-3.5-fold and decreased glucose by 40-50% in both diet groups, consistent with similar anaerobic and aerobic metabolism in both diet groups during high hippocampal activity. Overall, these data (1) defined the BHB concentration in the hippocampal extracellular fluid in KD-fed mice and (2) showed lower glucose metabolism compared to control diet-fed mice. This work will now enable other researchers to mimic the hippocampal extracellular environment in experiments aimed at deciphering the mechanisms of the KD.     

Diethyldithiocarbamate, copper and neurological disorders.

Diethyldithiocarbamate (DEDTC) given orally to rats without any addition of copper considerably increased the concentration of Cu in the brain without any change in the other tested tissues. Cysteine, comparatively studied, did not induce any change in the brain Cu level. Based on these findings and the literature data concerning DEDTC effects in animal and human, we put forward the hypothesis that the main effect of DEDTC is to provoke in the brain not a deficiency but an excess of Cu liberated from the lipophilic complex Cu-DEDTC. Cu is then engaged in an oscillatory oxido reduction giving a Cu++ cation radical able to induce deleterious effects on tissues in a similar way as paraquat. The practical consequences of this hypothesis are considered.     

Feeding behavior, rumination, reticulo-ruminal fermentation and plasma volatile fatty acids, compared in goats and sheep; influence of the diet

Two-year old Alpine she-goats (n = 3) and Texel ewes (n = 3) were compared as to eating behaviour, rumination and volatile fatty acids (VFA) in rumen and blood. The animals were fed once daily with two different proportions (20 and 80%) of barley and hay. Dry matter intake was fixed at 48 g D.M/kg P0.75 per day. In similar feeding and environmental conditions, eating behaviour and rumination of goats and sheep did not differ much: the goats tended to eat faster and there were more rumination periods in the sheep. Latency time, mean duration of rumination periods, daily rumination time and circadian pattern of rumination did not differ significantly between the two species (fig. 1). With both diets we observed a higher VFA concentration and a lower acetate/propionate ratio in the rumen of the ewes; however, rumen pH was lower only in those eating the 80% barley ration (fig. 2). Blood VFA in the jugular vein did not differ between sheep and goats (fig. 3). The proportion of cereals and hay in the diets affected rumen fermentation and rumination pattern in both species. With a higher concentrate/roughage ratio, rumen and plasma VFA increased, while the pH and acetate/propionate ratio in the lumen juice, the number of rumination periods and daily rumination time decreased. When the animals were fed the 80% barley ration, there was practically no rumination in the first 9 h after the single meal. During this time, rumen pH was minimal and VFA levels in the rumen and blood were maximal.     

Unusual fatty acids in turkeys fed a diet containing "Toprina". I. Composition of the higher fatty acids in "Toprina", the diet, feces and blood]

Totals lipids (TL) phospholipids (PL) and fatty acid composition of TL and PL (the latter in blood only) were estimated in Candida lipolytica cultivated on n-alkanes by industrial method ("Toprina"), in 0%, 10% and 15% "Toprina" diets and in faeces and blood of turkeys, males and females, fed these diets. With increasing concentrations of dietary "Toprina" phospholipids and 15:0 and 17:0 fatty acids increased, 17:1 and 17:2, typical of the product, appeared and increased in diets and blood. Other lipid and fatty acids values were not affected by dietary yeast.     

Antioxidants, oxidative stress, and degenerative neurological disorders

Recently, clinical trials of several neurodegenerative diseases have increasingly targeted the evaluation of the effectiveness of various antioxidants. The results so far are encouraging but variable and thus confusing. Rationale for the possible clinical effectiveness of antioxidants in several degenerative conditions has arisen out of the many years of basic science generally showing that reactive oxygen species (ROS) and oxidative damage are important factors in the processes involved. Aging is one of the most significant risk factors for degenerative neurological disorders. Basic science efforts in our laboratory have centered on exploring the role of ROS and oxidative stress in neurodegenerative processes. The present review brings together some of the basic concepts we have learned by following this approach for the last 20 years and specifically the results we have obtained by following up on our serendipitous findings that a nitrone-based free radical trap, alpha-phenyl-tert-butylnitrone (PBN), has neuroprotective activity in several experimental neurodegenerative models. The mechanistic basis of the neuroprotective activity of PBN does not appear to rely on its general free radical trapping or antioxidant activity per se, but its activity in mediating the suppression of genes induced by pro-inflammatory cytokines and other mediators associated with enhanced neuroinflammatory processes. Neuroinflammatory processes, induced in part by pro-inflammatory cytokines, yield enhanced ROS and reactive nitric oxide species (RNS) as well as other unknown components that have neurotoxic properties. Neurotoxic amounts of RNS are formed by the activity of inducible nitric oxide synthase (iNOS). The demonstration of enhanced 3-nitro-tyrosine formation in affected regions of the Alzheimer's brain, in comparison to age-matched controls, reinforces the importance of neuroinflammatory processes. iNOS induction involves activation by phosphorylation of the MAP kinase p38 and can be induced in cultured astrocytes by IL-1beta or H2O2. The action of PBN and N-acetyl cysteine to suppress the activation of p38 was demonstrated in cultured astrocytes. The demonstration of activated p38 in neurons surrounding amyloid plaques in affected regions of the Alzheimer's brain attest to enhanced signal transduction processes in this neurodegenerative condition. The major themes of ROS and RNS formation associated with neuroinflammation processes and the suppression of these processes by antioxidants and PBN continue to yield promising leads for new therapies. Outcomes of clinical trials on antioxidants will become less confusing as more knowledge is amassed on the basic processes involved.     

Molecular recognition of fatty acids by peroxisome proliferator-activated receptors

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors for fatty acids (FAs) that regulate glucose and lipid homeostasis. We report the crystal structure of the PPAR delta ligand-binding domain (LBD) bound to either the FA eicosapentaenoic acid (EPA) or the synthetic fibrate GW2433. The carboxylic acids of EPA and GW2433 interact directly with the activation function 2 (AF-2) helix. The hydrophobic tail of EPA adopts two distinct conformations within the large hydrophobic cavity. GW2433 occupies essentially the same space as EPA bound in both conformations. These structures provide molecular insight into the propensity for PPARs to interact with a variety of synthetic and natural compounds, including FAs that vary in both chain length and degree of saturation.     

Differential modulation of Toll-like receptors by fatty acids: preferential inhibition by n-3 polyunsaturated fatty acids

Human subjects consuming fish oil showed a significant suppression of cyclooxygenase-2 (COX-2) expression in blood monocytes when stimulated in vitro with lipopolysaccharide (LPS), an agonist for Toll-like receptor 4 (TLR4). Results with a murine monocytic cell line (RAW 264.7) stably transfected with COX-2 promoter reporter gene also demonstrated that LPS-induced COX-2 expression was preferentially inhibited by docosahexaenoic acid (DHA, C22:6n-3) and eicosapentaenoic acid (EPA, C20:5n-3), the major n-3 polyunsaturated fatty acids (PUFAs) present in fish oil. Additionally, DHA and EPA significantly suppressed COX-2 expression induced by a synthetic lipopeptide, a TLR2 agonist. These results correlated with the preferential suppression of LPS- or lipopeptide-induced NF kappa B activation by DHA and EPA. The target of inhibition by DHA is TLR itself or its associated molecules, but not downstream signaling components. In contrast, COX-2 expression by TLR2 or TRL4 agonist was potentiated by lauric acid, a saturated fatty acid. These results demonstrate that inhibition of COX-2 expression by n-3 PUFAs is mediated through the modulation of TLR-mediated signaling pathways. Thus, the beneficial or detrimental effects of different types of dietary fatty acids on the risk of the development of many chronic inflammatory diseases may be in part mediated through the modulation of TLRs.