Biohydrogenation of C20 polyunsaturated fatty acids by anaerobic bacteria

The PUFAs include many bioactive lipids. The microbial metabolism of C₁₈ PUFAs is known to produce their bioactive isomers, such as conjugated FAs and hydroxy FAs, but there is little information on that of C₂₀ PUFAs. In this study, we aimed to obtain anaerobic bacteria with the ability to produce novel PUFAs from C₂₀ PUFAs. Through the screening of ∼100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and EPA (cis-5,cis-8,cis-11,cis-14,cis-17-EPA) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of a bacterium transforming C₂₀ PUFAs into corresponding non-methylene-interrupted FAs. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at the ω6 and ω9 positions in various C₁₈ and C₂₀ PUFAs into a trans double bond at the ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.     

Dietary polyunsaturated fatty acids improve cholinergic transmission in the aged brain

The cholinergic theory of aging states that dysfunction of cholinergic neurons arising from the basal forebrain and terminating in the cortex and hippocampus may be involved in the cognitive decline that occurs during aging and Alzheimer’s disease. Despite years of research, pharmacological interventions to treat or forestall the development of Alzheimer’s disease have primarily focused on enhancing cholinergic transmission, either through increasing acetylcholine (ACh) synthesis or inhibition of the acetylcholinesterase enzyme responsible for ACh hydrolysis. However, recent studies have indicated that dietary supplementation can impact the cholinergic system, particularly during aging. The purpose of the present review is to examine the relevant research suggesting that cholinergic functioning may be maintained during aging via consuming a diet containing polyunsaturated fatty acids (PUFAs). The data reviewed herein indicate that, at least in animal studies, inclusion of PUFAs in the diet can improve cholinergic transmission in the brain, possibly leading to improvements in cognitive functioning.     

The inhibitory effect of polyunsaturated fatty acids on human CYP enzymes

The inhibitory effect of saturated fatty acids (SFAs): palmitic acid (PA), stearic acid (SA) and polyunsaturated fatty acids (PUFAs): linoleic acid (LA), linolenic acid (LN), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on six human drug-metabolizing enzymes (CYP1A2, 2C9, 2C19, 2D6, 2E1 and 3A4) was studied. Supersomes from baculovirus-expressing single isoforms were used as the enzyme source. Phenacetin O-deethylation (CYP1A2), diclofenac 4-hydroxylation (CYP2C9), mephenytoin 4-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1) and midazolam 1-hydroxylation (CYP3A4) were used as the probes. Results show that all the five examined PUFAs competitively inhibited CYP2C9- and CYP2C19-catalyzed metabolic reactions, with Ki values ranging from 1.7 to 4.7 microM and 2.3 to 7.4 microM, respectively. Among these, AA, EPA and DHA tended to have greater inhibitory potencies (lower IC(50) and Ki values) than LA and LN. In addition, these five PUFAs also competitively inhibited the metabolic reactions catalyzed by CYP1A2, 2E1 and 3A4 to a lesser extent (Ki values>10 microM). On the other hand, palmitic and stearic acids, the saturated fatty acids, had no inhibitory effect on the activities of six human CYP isozymes at concentrations up to 200 microM. Incubation of PUFAs with CYP2C9 or CYP2C19 in the presence of NADPH resulted in the decrease of PUFA concentrations in the incubation mixtures. These results indicate that the PUFAs are potent inhibitors as well as the substrates of CYP2C9 and CYP2C19.     

Inhibition of FASN reduces the synthesis of medium-chain fatty acids in goat mammary gland

Fatty acid synthase (FASN) is known as a crucial enzyme of cellular de novo fatty acid synthesis in mammary gland which has been proved as the main source of short and medium-chain fatty acids of milk. However, the regulatory role of FASN in goat-specific milk fatty acids composition remains unclear. We cloned and analyzed the full-length of FASN gene from the mammary gland of Capra hircus (Xinong Saanen dairy goat) (DQ 915966). Comparative gene expression analysis suggested that FASN is predominantly expressed in fat, small intestine and mammary gland tissues, and expresses higher level at lactation period. Inhibition of FASN activity by different concentrations (0, 5, 15, 25 and 35 μM) of orlistat, a natural inhibitor of FASN, resulted in decreased expression of acetyl-CoA carboxylase α (ACCα), lipoprotein lipase and heart-type fatty acid binding protein (H-FABP) in a concentration-dependent manner in goat mammary gland epithelial cells (GMEC). Similar results were also obtained by silencing of FASN. Additionally, reduction of FASN expression also led to apparent decline of the relative content of decanoic acid (C10:0) and lauric acid (C12:0) in GMEC. Our study provides a direct evidence for inhibition of FASN reduces cellular medium-chain fatty acids synthesis in GMEC.     

Dependence on the phospholipid polyunsaturated fatty acids of the oxidative injury of isolated cardiomyocytes

We investigated the influence of PUFA in phospholipids (PL) on the functional characteristics of cultured cardiomyocytes (CM) in basal conditions and during free radical (FR) stress provoked either by the xanthine/xanthine oxidase (X/XO) system or by a (9Z, 11E, 13 (S), 15Z)-13-hydroperoxyoctadecatrienoic acid (13-HpOTrE). CM were grown in media containing either n - 3 (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA) or n - 6 (arachidonic acid, AA). These two groups of CM displayed different PUFA n - 6/n - 3 ratio in PL. However, their basal electromechanical characteristics were similar. The X/XO system drastically altered CM functions, without difference between the two groups of CM. 13-HpOTrE caused a moderate and reversible depression in action potential parameters, which was dependent upon the PL PUFA, since the n - 3-enriched CM exhibited an earlier functional depression but faster recovery. Thus, the peroxidative damage of CM depended on a cross relationship between FR species and the PL PUFA composition.     

Identification of unusual fatty acids of four alpine plant species from the Pamirs

Fatty acid composition and structure in total lipids from the green above-ground parts of four alpine plants, Oxygraphis glacialis, Primula macrophylla, Rhodiola pamiroalaica, and Swertia marginata, were established by GC and GC-MS. A total of 55 fatty acids was detected, and 48 of them were identified. Ubiquitous palmitate, linoleate, and linolenate predominated in the lipids accounting for about 72-90% of the total fatty acids. At the same time, the latter contained numerous species, which were unusual for higher plants and included saturated odd-numbered n-acids (six C15-C25 species, 0.26-1.40%), saturated even-numbered very-long-chain n-acids (six C20-C30 species, 1.00-2.49%), iso-acids (nine C15-C26 species, 0.64-1.53%), anteiso-acids (four C15-C20 species, 0.08-1.57%), certain uncommon mono- and dienoic acids, as well as 16:3omega3, 18:3omega6, and 18:4omega3 acids that are absent from the most higher plants. Nine fatty acids were found here for the first time in higher plants and two may be new to science. The evidence on the unusual fatty acids is discussed with respect to their distribution in living organisms, pathways of biosynthesis, and chemotaxonomic role.     

A review of nutritional effects on fat composition of animal products with special emphasis on n-3 polyunsaturated fatty acids

The fatty acid composition of animal products (eggs, milk and meat) is the reflect of both the tissue fatty acid biosynthesis and the fatty acid composition of ingested lipids. This relationship is stronger in monogastrics (pigs, poultry and rabbits) than in ruminants, where dietary fatty acids are hydrogenated in the rumen. There is an increasing recognition of the health benefits of polyunsaturated fatty acids (PUFA), because these fatty acids are essential for humans. In addition, the ratio n-6/n-3 fatty acids in the human diet is important. This ratio by far exceeds the recommended value of 5. Therefore, inclusion of fish meals, or n-3 PUFA rich oils, or linseed in animal diets is a valid means of meeting consumer demand for animal products that are nutritionally beneficial.The studies that are undertaken on animals mainly use diets supplemented with linseed, as a source of n-3 fatty acids. The use of linseed diets generally leads to an increased n-3 fatty acid content in animal products (egg, meat, milk) in ruminants and monogastrics. Recent studies have also demonstrated that neither the processing nor the cooking affects the PUFA content of pork meat or meat products.The ability of unsaturated fatty acids, especially those with more than two double bonds, to rapidly oxidise, is important in regulating the shelf life of animal products (rancidity and colour deterioration); however, a good way to avoid such problems is to use antioxidant products (such as vitamin E) in the diet.Some studies also show that it is not necessary to feed animals with linseed-supplemented diets for a long time to have the highest increase in PUFA content of the products. So, short-term diet manipulation can be a practical reality for industry.As the market for n-3 PUFA enriched products is today limited in most countries, other studies must be undertaken to develop this kind of production.     

The predominance of polyunsaturated fatty acids in the butterfly Morpho peleides before and after metamorphosis

We hypothesized that the polyunsaturated fatty acids of the butterfly were probably derived from the diet and that there might be a great loss of body fat during metamorphosis. To substantiate these hypotheses, we analyzed the fatty acid composition and content of the diet, the larva, and the butterfly Morpho peleides. Both the diet and the tissues of the larva and butterfly had a high concentration of polyunsaturated fatty acids. In the diet, linolenic acid accounted for 19% and linoleic acid for 8% of total fatty acids. In the larva, almost 60% of the total fatty acids were polyunsaturated: linolenic acid predominated at 42% of total fatty acids, and linoleic acid was at 17%. In the butterfly, linolenic acid represented 36% and linoleic acid represented 11% of total fatty acids. The larva had a much higher total fatty acid content than the butterfly (20.2 vs. 6.9 mg). Our data indicate that the transformation from larva to butterfly during metamorphosis drastically decreased the total fatty acid content. There was bioenhancement of polyunsaturated fatty acids from the diet to the larva and butterfly. This polyunsaturation of membranes may have functional importance in providing membrane fluidity useful in flight.     

Effect of essential fatty acids on circulating T cell subsets in patients with colorectal cancer

The effect of essential fatty acids (EFA), given orally as dietary supplements, on the responsiveness in vitro of peripheral blood lymphocytes (PBL), to the mitogen concanavalin A have been studied in 10 patients with localized and 14 patients with advanced colorectal cancer. The degree of lymphocyte activation was assessed by measuring the amount of tritiated [³H]thymidine incorporated into newly synthesised lymphocyte DNA. The results were expressed as stimulation indices. T cell responses to concanavalin A stimulation showed a significant reduction of stimulation indices following EFA supplementation, in both the localized (P=0.026) and advanced (P=0.016) tumour groups, when compared with pretreatment activity in vitro. Mixing experiments, using EFA-supplemented and non-EFA-supplemented lymphocytes with concanavalin A, suggest no enhancement of T suppressor cell activity. Cell surface marker analysis (fluorescence-activated cell sorting for CD phenotyping) revealed a reduction of absolute numbers of CD4⁺ and CD8⁺ lymphocytes following EFA supplementation. The stimulation indices returned to presupplementation values 3 months following cessation of EFA intake. There was no significant change of these indices in the control (no EFA supplementation) advanced tumour group tested. This study suggests that EFA supplementation in patients with colorectal cancer selectively reduces circulating PBL. and T cell subset (including suppressor cells) numbers and/or activity. Such effects may have an important outcome in patients with malignant disease.This work was supported by grants from the Grampian Health Broad, the Royal College of Surgeons of Edinburgh, and Scottish Hospital Endownment Research Trust     

Methoxylated fatty acids in Blumeria graminis conidia

The total fatty acids (FA) composition of Blumeria graminis f.sp. tritici conidia, the causal agent of wheat powdery mildew, was analyzed as a function of their age. A total of 19 FA (C12-C24 saturated and unsaturated) and unusual methoxylated fatty acids (mFA) were detected in young, intermediate and old conidia. Two very long chain methoxylated FA were identified by GC-MS as 3-methoxydocosanoic and 3-methoxytetracosanoic acids. Medium chain FA were predominant in young conidia (75%, including 13% of mFA) while very long chain fatty acids constituted the major compounds in old conidia (74%, including 30% of mFA). We have shown for the first time that the total FA composition is strongly correlated with the age of B. graminis f.sp. tritici (Bgt) conidia.     

四个脂肪酸合成酶基因在哺乳动物细胞中的超表达提高长链多不饱和脂肪酸生物合成效率

DHA(22:6n-3)、EPA(20:5n-3)和ARA(20:4n-6)三种长链多不饱和脂肪酸在生物体内活性最强,它们在促进大脑发育和功能维持以及在预防和治疗心血管疾病、炎症、癌症等多种疾病方面有着重要作用。然而,尽管哺乳动物体内有完整的长链多不饱和脂肪酸合成酶系,但哺乳动物合成这些长链多不饱和脂肪酸的效率很低而主要依赖于食物获取。本研究应用转基因方法,将哺乳动物来源的Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶这4种酶的编码基因构建成为一个多基因表达载体,然后转染哺乳动物细胞HEK293T,实现了4个目的基因的超表达,再通过气质联用(GC-MS)分析证实了DHA、EPA和ARA等长链多不饱和脂肪酸的合成效率及水平显著增加,DHA的水平更是提高了2.5倍。由此可见,哺乳动物具有某种抑制长链多不饱和脂肪酸高水平合成的机制,但通过Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶的超表达,能够打破哺乳动物这种抑制机制,从而显著提高DHA、EPA、ARA等的合成水平。同时,本研究的思路也为在转基因动物中生产长链多不饱和脂肪酸提供了重要的启示。     

Colimitation of a freshwater herbivore by sterols and polyunsaturated fatty acids

Empirical data providing evidence for a colimitation of an herbivore by two or more essential nutrients are scarce, particularly in regard to biochemical resources. Here, a graphical model is presented, which describes the growth of an herbivore in a system with two potentially limiting resources. To verify this model, life-history experiments were conducted with the herbivore Daphnia magna feeding on the picocyanobacterium Synechococcus elongatus, which was supplemented with increasing amounts of cholesterol either in the presence or the absence of saturating amounts of eicosapentaenoic acid (EPA). For comparison, D. magna was raised on diets containing different proportions of S. elongatus and the cholesterol- and EPA-rich eukaryotic alga Nannochloropsis limnetica. Somatic and population growth of D. magna on a sterol- and EPA-deficient diet was initially constrained by the absence of sterols. With increased sterol availability, a colimitation by EPA became apparent and when the sterol requirements were met, the growth-limiting factor was shifted from a limitation by sterols to a limitation by EPA. These data imply that herbivores are frequently limited by two or more essential nutrients simultaneously. Hence, the concept of colimitation has to be incorporated into models assessing nutrient-limited growth kinetics of herbivores to accurately predict demographic changes and population dynamics.     

The effect of bio-converted polyunsaturated fatty acids on the oxidation of TAG containing highly unsaturated fatty acids

Microbial modification of vegetable fatty acids can often lead to special changes in their structure and in biological function. A bacterial strain, Pseudomonas aeruginosa PR3, is known to carry out multiple hydroxylations on polyunsaturated fatty acids containing 1,4-cis, cis diene structural units, resulting in antibacterial activity. In this paper, in an effort to understand the overall mechanism involved in the varied biological functions of the complicated metabolites of bio-converted polyunsaturated fatty acids, we performed bioconversion of several polyunsaturated fatty acids using PR3, and determined their oxidative activities against fish oil. Bio-converted linoleic acid, eicosapentanoic acid, and docosahexanoic acid promoted effectively oxidation of fish oil. It is assumed that this oxidative effect could plausibly play an important role in the antimicrobial function of these bio-converted fatty acids.     

Role of omega-3 polyunsaturated fatty acids on cyclooxygenase-2 metabolism in brain-metastatic melanoma

Cyclooxygenase-2 (COX-2) is important in the progression of epithelial tumors. Evidence indicates that omega-6 PUFAs such as arachidonic acid (AA) promote the growth of tumor cells; however, omega-3 fatty acids [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] inhibit tumor cell proliferation. We investigated the effects of omega-3 PUFA on the expression and function of COX-2 in 70W, a human melanoma cell line that metastasizes to the brain in nude mice. We show that 1) tumor necrosis factor-alpha upregulates the expression of both COX-2 mRNA and prostaglandin E2 (PGE2) production, and 2) omega-3 and omega-6 PUFA regulate COX-2 mRNA expression and PGE2 production. AA increased COX-2 mRNA expression and prostaglandin production in omega-6-stimulated 70W cells. Conversely, COX-2 mRNA expression decreased in cells incubated with EPA or DHA. AA increased Matrigel invasion 2.4-fold, whereas EPA or DHA did not. Additionally, PGE2 increased in vitro invasion 2.5-fold, whereas exposure to PGE3 significantly decreased invasion. Our results demonstrate that incubation of 70W cells with either AA or PGE2 increased invasiveness, whereas incubation with EPA or DHA downregulated both COX-2 mRNA and protein expression, with a subsequent decrease in Matrigel invasion. Taken together, these results indicate that omega-3 PUFA regulate COX-2-mediated invasion in brain-metastatic melanoma.     

Anti-inflammatory effects of polyunsaturated fatty acids in THP-1 cells

The effects of linoleic acid (LA), alpha-linolenic acid (ALA), and docosahexaenoic acid (DHA) were compared to that of palmitic acid (PA), on inflammatory responses in human monocytic THP-1 cells. When cells were pre-incubated with fatty acids for 2-h and then stimulated with lipopolysaccharide for 24-h in the presence of fatty acids, secretion of interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha (TNFalpha) was significantly decreased after treatment with LA, ALA, and DHA versus PA (P < 0.01 for all); ALA and DHA elicited more favorable effects. These effects were comparable to those for 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and were dose-dependent. In addition, LA, ALA, and DHA decreased IL-6, IL-1beta, and TNFalpha gene expression (P < 0.05 for all) and nuclear factor (NF)-kappaB DNA-binding activity, whereas peroxisome proliferator-activated receptor-gamma (PPARgamma) DNA-binding activity was increased. The results indicate that the anti-inflammatory effects of polyunsaturated fatty acids may be, in part, due to the inhibition of NF-kappaB activation via activation of PPARgamma.     

Synthesis of trans unsaturated fatty acids in Pseudomonas putida P8 by direct isomerization of the double bond of lipids

The phospholipids of Pseudomonas putida P8 contain monounsaturated fatty acids in the cis and trans configuration. Cells of this phenol-degrading bacterium change the proportions of these isomers in response to the addition or elimination of a membrane active compound such as 4-chlorophenol. This study undoubtedly reveals that the cis unsaturated fatty acids are directly converted into trans isomers without involvement of de novo synthesis of fatty acids. Oleic acid, which cannot be synthesized by this bacterium, was incorporated as a cis unsaturated fatty acid marker in the membrane lipids of growing cells. The conversion of this fatty acid into the corresponding trans isomer was demonstrated by gas chromatographic-mass spectrometric analysis and use of ¹⁴C-labeled oleic acid. Separation and isolation of the cellular membranes showed that the fatty acid isomerase is located in the cytoplasmic membrane of P. putida P8.Abbreviation 4-CP 4-chlorophenol     

Glucose transport and utilization are altered in the brain of rats deficient in n-3 polyunsaturated fatty acids

Long-chain polyunsaturated (n-3) fatty acids have been reported to influence the efficiency of membrane receptors, transporters and enzymes. Because the brain is particularly rich in docosahexaenoic acid (DHA, 22:6 n-3), the present study addresses the question of whether the 22:6 n-3 fatty acid deficiency induces disorder in regulation of energy metabolism in the CNS. Three brain regions that share a high rate of energy metabolism were studied: fronto-parietal cortex, hippocampus and suprachiasmatic nucleus. The effect of the diet deficient in n-3 fatty acids resulted in a 30-50% decrease in DHA in membrane phospholipids. Moreover, a 30% decrease in glucose uptake and a 20-40% decrease in cytochrome oxidase activity were observed in the three brain regions. The n-3 deficient diet also altered the immunoreactivity of glucose transporters, namely GLUT1 in endothelial cells and GLUT3 in neurones. In n-3 fatty acid deficient rats, GLUT1-immunoreactivity readily detectable in microvessels became sparse, whereas the number of GLUT3 immunoreactive neurones was increased. However, western blot analysis showed no significant difference in GLUT1 and GLUT3 protein levels between rats deficient in n-3 fatty acids and control rats. The present results suggest that changes in energy metabolism induced by n-3 deficiency could result from functional alteration in glucose transporters.     

Effect of dietary polyunsaturated fatty acids on reproductive output and larval growth of bivalves

The pre-spawning condition of adult bivalves is influenced by quantity and quality of available food. For bivalves, the essential polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA) 20:5(n-3) and docosahexaenoic acid (DHA) 22:6(n-3) are presumed to determine the nutritional value of algae. Macoma balthica kept on a broodstock diet supplemented with PUFAs spawned a larger number of eggs (average 22220) per female and larger sized eggs (106.7 μm) compared to adults kept on a diet without PUFA supplementation (962.5 eggs with average size 99.8 μm).Larvae of M. balthica from the same parental pool however did not profit from a diet where a part was replaced with PUFA spheres. Instead, larvae reared on Isochrysis sp. showed lower mortality and higher growth rates than larvae fed on the same algae supplemented with lipid spheres. Crassostrea gigas larvae showed no clear response to a PUFA supplemented diet.