Relation between phylogenetic position, lipid metabolism and butyrate production by different Butyrivibrio-like bacteria from the rumen

The Butyrivibrio group comprises Butyrivibrio fibrisolvens and related Gram-positive bacteria isolated mainly from the rumen of cattle and sheep. The aim of this study was to investigate phenotypic characteristics that discriminate between different phylotypes. The phylogenetic position, derived from 16S rDNA sequence data, of 45 isolates from different species and different countries was compared with their fermentation products, mechanism of butyrate formation, lipid metabolism and sensitivity to growth inhibition by linoleic acid (LA). Three clear sub-groups were evident, both phylogenetically and metabolically. Group VA1 typified most Butyrivibrio and Pseudobutyrivibrio isolates, while Groups VA2 and SA comprised Butyrivibrio hungatei and Clostridium proteoclasticum, respectively. All produced butyrate but strains of group VA1 had a butyrate kinase activity <40 U (mg protein)⁻¹, while strains in groups VA2 and SA all exhibited activities >600 U (mg protein)⁻¹. The butyrate kinase gene was present in all VA2 and SA bacteria tested but not in strains of group VA1, all of which were positive for the butyryl-CoA CoA-transferase gene. None of the bacteria tested possessed both genes. Lipase activity, measured by tributyrin hydrolysis, was high in group VA2 and SA strains and low in Group VA1 strains. Only the SA group formed stearic acid from LA. Linoleate isomerase activity, on the other hand, did not correspond with phylogenetic position. Group VA1 bacteria all grew in the presence of 200 μg LA ml⁻¹, while members of Groups VA2 and SA were inhibited by lower concentrations, some as low as 5 μg ml⁻¹. This information provides strong links between phenotypic and phylogenetic properties of this group of clostridial cluster XIVa Gram-positive bacteria.     

Genetic control of polyunsaturated fatty acid biosynthesis in flax (Linum usitatissimum) seed oil

Summary The inheritance of two mutants of flax (Linum usitatissimum), having altered proportions of the C18 polyunsaturated fatty acids, linoleic and linolenic, was examined. Both lines, M1589 and M1722, are homozygous for a single gene mutation which reduces linolenic acid content from 34% to 22% and raises linoleic acid from 15% to 27%. Genetic analysis of crosses involving M1589, M1722 and their parental cultivar Glenelg revealed that these mutations are in different unlinked genes and exhibit additive (codominant) gene action. The symbolsLn1 andLn2 are proposed for the mutated genes in M1589 and M1722, respectively. Recombinant genotypes homozygous for the mutant alleles at both loci are very low in linolenic acid (2%) and high in linoleic acid (48%), with unaltered proportions of other fatty acids. The complete inverse correlation between linoleic and linolenic acids (r=-0.98) indicates that the mutations block the synthesis of linolenic acid at the linoleic desaturation step.     

Exploring the relationship between bacterial genera and lipid metabolism in bovine rumen

The rumen is characterised by a complex microbial ecosystem, which is particularly active in lipid metabolism. Several studies demonstrated a role of diet and breed on bacterial community profile, with the effect on metabolic pathways. Despite the knowledge achieved on metabolism and the bacterial profile, little is known about the relationship between individual bacteria and metabolic pathways. Therefore, a multivariate approach was used to search for possible relationships between bacteria and products of several pathways. The correlation between rumen bacterial community composition and rumen lipid metabolism was assessed in 40 beef steers (20 Maremmana and 20 Aubrac) reared with the same system and fed the same diet. A canonical discriminant analysis combined with a canonical correlation analysis (CCA) was performed to explore this correlation. The variables showing a Pearson correlation higher than 0.6 as absolute value and significant were retained for CCA considering the relationship of bacterial composition with several metabolic pathways. The results indicated that some bacterial genera could have significant impacts on the presence of several fatty acids. However, the relationship between genera and fatty acid changes according to the breed, demonstrating that the metabolic pathways change according to the host genetic background, related to breed evolution, although there is also an intra-breed genetic background which should not be ignored. In Maremmana, Succiniclasticum and Rikenellaceae_RC9_gut_group showed a high positive correlation with dimethylacetals (DMAs) DMA_C13:0, DMA_C14:0, DMA_C14:0iso, DMA_C15:0, DMA_C15:0iso, and DMA_C18:0. Prevotellaceae_UCG-003 correlates with C18:3c9c12c15 and C18:1t11, while Fibrobacter and Succiniclasticum correlate with C18:2c9t11 and Lachnospiraceae_NK3A20_group correlates with C18:1c12. Prevotellaceae_UCG-003, Ruminococcaceae_UCG-010, and Oribacterium showed a positive correlation with C13:0iso, and C17:0. Conversely, in Aubrac, Treponema_2 and Rikenellaceae_RC9_gut_group correlated with DMA_C14:0iso, DMA_C16:0iso, DMA_C17:0iso, while Ruminococcaceae_UCG-010, Christensenellaceae_R-7_group and Ruminococcaceae_NK4A214_group correlated with DMA_C18:1t11, DMA_C14:0, DMA_C18:1c12. Acetitomaculum correlated with C18:2c9c12, C18:1c12, C18:1c13, C18:1t12 and Lachnospiraceae_NK3A20_group with C18:1t6-8 and C18:1t9. Saccharofermentas, Ruminococcaceae_UCG-010 and Rikenellaceae_RC9_gut_group correlated with C18:2c9t11 while, Prevotellaceae_UCG-001 and Ruminococcus_1 correlated with C14:0iso, C15:0, C15:0iso, C17:0. Saccharofermentans, Rikenellaceae_RC9_gut_group, Ruminococcaceae_NK4A214_group, and Ruminococcaceae_UCG-010 correlated with C13:1c12 and C16:0iso. These results lead to hypothesise a possible association between several metabolic pathways and one or a few bacterial genera. If these associations are confirmed by further investigations that verify the causality of a bacterial genus with a particular metabolic process, it will be possible to deepen the knowledge on the activity of the rumen population in lipid metabolism. This approach appears to be a promising tool for uncovering the correlation between bacterial genera and products of rumen lipid metabolism.     

Enzymatic hydrolysis of soybean oil using lipase from different sources to yield concentrated of polyunsaturated fatty acids

The ability of three commercially available lipases to mediate the hydrolysis of the soybean oil to yield concentrated of essential fatty acids was evaluated. The tested lipases were from microbial (Candida rugosa and Thermomyces lanuginosa) and animal cells (Porcine pancreatic lipase). In terms of free fatty acids, microbial lipases were more effective to promote the enzymatic hydrolysis of the soybean oil (over 70%) than the porcine pancreatic lipase (24%). In spite of this, porcine pancreatic lipase (PPL) showed the most satisfactory specificity towards both essential fatty acids and was, therefore, chosen to carry out additional studies. An experimental design was performed taking into consideration the enzyme and NaCl amounts as independent variables. The main effects were fitted by multiple regression analysis to a linear model and maximum fatty acids concentration could be obtained using 3.0 wt% of lipase and 0.08 wt% of NaCl. The mathematical model representing the hydrolysis degree was found to describe adequately the experimental results. Under these conditions, concentrations of 29.5 g/L and 4.6 g/L for linoleic and linolenic acids, respectively, were obtained.     

Correlations between gametophytic (pollen) and sporophytic (seed) generations for polyunsaturated fatty acids in oilseed rape Brassica napus L.

Summary Lipids were extracted from the diploid seed and haploid pollen of Brassica napus L. Two fractions of pollen lipids, namely the diploid-specified pollen-coat and the haploid-specified internal cytoplasmic lipids were obtained. Significant correlations exist between pollen and seed generations for linoleic (182) and linolenic (183) acids. In pollen internal storage lipids, the level of 183 is positively correlated and the level of 182 is negatively correlated with the level of 183 in seed lipids. Evidence is presented that strongly supports the hypothesis that lipid biosynthesis occurs within the pollen and that synthesis is specified by haploid genes. These data support the concept of pollen selection, so that selecting among living pollen grains for superior individuals has potential as a new plant breeding tool for improving seed oil quality.     

山楂蜂花粉中脂肪酸的GC-MS分析

用乙醇-氯仿作溶剂,提取出山楂蜂花粉粗脂肪,然后进行甲酯化处理,用气相色谱/质谱联用技术进行分离鉴定,共鉴定出10种脂肪酸,并测定相对含量,多不饱和脂肪酸含量较高,其中亚油酸相对含量为47.84%,亚麻酸相对含量为19.30%.     

Modulation of the mitochondrial large-conductance calcium-regulated potassium channel by polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) and their metabolites can modulate several biochemical processes in the cell and thus prevent various diseases. PUFAs have a number of cellular targets, including membrane proteins. They can interact with plasma membrane and intracellular potassium channels. The goal of this work was to verify the interaction between PUFAs and the most common and intensively studied mitochondrial large conductance Ca²⁺-regulated potassium channel (mitoBK_Ca). For this purpose human astrocytoma U87 MG cell lines were investigated using a patch-clamp technique. We analyzed the effects of arachidonic acid (AA); eicosatetraynoic acid (ETYA), which is a non-metabolizable analog of AA; docosahexaenoic acid (DHA); and eicosapentaenoic acid (EPA). The open probability (P_o) of the channel did not change significantly after application of 10 μM ETYA. P_o increased, however, after adding 10 μM AA. The application of 30 μM DHA or 10 μM EPA also increased the P_o of the channel. Additionally, the number of open channels in the patch increased in the presence of 30 μM EPA. Collectively, our results indicate that PUFAs regulate the BK_Ca channel from the inner mitochondrial membrane.     

Application of high-performance liquid chromatography of plasma fatty acids as their phenacyl esters to evaluate splanchnic and renal fatty acid balance in vivo

Plasma fatty acids from renal and hepatic veins, and arterialized hand vein obtained in 20 subjects before and after insulin infusion were separated by reversed-phase high-performance liquid chromatography following phenacyl esterification. Separation and quantification over the range 1.0–100 nmol per injection of nine fatty acids was achieved within 60 min using [²H₃₁]palmitic acid as internal standard. Analytical recoveries were greater than 90% and the intra- and inter-assay coefficients of variation were less than 2.5 and 4.0%, respectively. Following insulin infusion, net splanchnic uptake of total fatty acids decreased from 3.0±0.3 to 1.0±0.1 μmol/kg min (p<0.01), whereas net renal balance remained neutral (−0.04±0.04 vs. −0.06±0.03 μmol/kg min, p=N.S.). Individual fatty acid balance varied from a low of 0.012±0.005 (myristic acid) to a high of 0.95±0.08 (oleic acid) μmol/kg min across the splanchnic tissues and from 0.005±0.002 (stearic acid) to 0.21±0.1 (oleic acid) μmol/kg min across the kidney. There is a substantial diversity in changes in plasma concentration and regional balance of individual fatty acid during short-term fasting and hyperinsulinemia. This method is simple, accurate, and can be applied to assess individual fatty acid metabolism in vivo.     

Anti-HCV activities of selective polyunsaturated fatty acids

HCV infection can lead to chronic infectious hepatitis disease with serious sequelae. Interferon-alpha, or its PEGylated form, plus ribavirin is the only treatment option to combat HCV. Alternative and more effective therapy is needed due to the severe side effects and unsatisfactory curing rate of the current therapy. In this study, we found that several polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) are able to exert anti-HCV activities using an HCV subgenomic RNA replicon system. The EC(50) (50% effective concentration to inhibit HCV replication) of AA was 4microM that falls in the range of physiologically relevant concentration. At 100microM, alpha-linolenic acid, gamma-linolenic, and linoleic acid only reduced HCV RNA levels slightly and saturated fatty acids including oleic acid, myristic acid, palmitic acid, and steric acid had no inhibitory activities toward HCV replication. When AA was combined with IFN-alpha, strong synergistic anti-HCV effect was observed as revealed by an isobologram analysis. It will be important to determine whether PUFAs can provide synergistic antiviral effects when given as food supplements during IFN-based anti-HCV therapy. Further elucidation of the exact anti-HCV mechanism caused by AA, DHA, and EPA may lead to the development of agents with potent activity against HCV or related viruses.     

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.     

Using microbial fatty acids to improve understanding of the contribution of solid associated bacteria to microbial mass in the rumen

This study sought to distinguish liquid-(LAB) and detached (SAB₁) and undetached (SAB₂) solid-associated bacteria through their fatty acid (FA) and purine base (PB) profiles. Fatty acids and PB were also evaluated as internal microbial markers for estimating microbial biomass associated with rumen particles. Four merino rams fitted with rumen cannulae and fed dehydrated alfalfa pellets provided rumen contents. In 3 consecutive weeks, rumen contents were collected and samples of LAB and SAB₁, total rumen content (TRC), washed rumen particles (WRP) and rumen particles after SAB₁ extraction (ERP) were obtained and analysed for PB and FA. The SAB₂ biomass composition was estimated from the non-NDF organic matter (OM) remaining in ERP. The concentration of total SAB biomass in particles was estimated using both PB and odd and branched-chain fatty acids (OBCFA). Concentrations of PB and OBCFA were highly correlated among the different rumen fractions. Marked differences between LAB and SAB populations occurred with LAB having higher PB content, lower FA content and a higher proportion (g/100 g fatty acids) of OBCFA than did SAB. The chemical composition of SAB₁ and SAB₂ was similar, except for the 15% higher crude protein content of the latter. The concentration of OBCFA (mg/g microbial OM) did not differ between bacterial fractions. The PB/OBCFA ratio (mg/mg) was higher in LAB (2.08) than in SAB (0.94). The ratio between branched-chain and odd-linear-chain FA was higher in LAB (2.26) than in SAB (1.46). Extraction of PB and OBCFA from WRP with our SAB detachment procedure was 61% and 31%, respectively. Estimated SAB₁ and total SAB biomass (mg OM/g WRP) were 158 and 266, and 47 and 164, respectively, using PB and OBCFA as microbial markers. This study suggests that the OBCFA have potential as internal microbial markers in rumen ecosystem studies.     

Metabolism of polyunsaturated fatty acids by larvae of the waxmoth,Galleria mellonella

The metabolic fates of linoleic (18:2n6) and linolenic (18:3n3) acids injected into the hemocoel of fifth instar larvae of the waxmoth, Galleria mellonella, were examined by radio-high-pressure liquid chromatography and radio-gas-liquid chromatography. In addition to undergoing β-oxidation and incorporation into neutral and phospholipid fractions, a portion of both of these C18 fatty acids was elongated and desaturated to longer chain and more unsaturated polyenoics. Radioactivity from linoleic acid was recovered in components that coeluted with 18:3, 18:4, 20:3, and 20:4. Radioactivity from linolenic acid was recovered in an unidentified component and in components that coeluted with 18:4, 20:3, and 20:5. Labeled arachidonic and eicosapentaenoic acids injected into waxmoth larvae were converted to prostaglandins, suggesting that one aspect of the biological significance of the elongation/desaturation reactions is to generate precursors for prostaglandin biosynthesis.     

Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools

BackgroundDietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations.ObjectiveTo evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, alters unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache.DesignSecondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3–L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet.ResultsCompared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3–L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations.ConclusionDietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFAs for 12 weeks further increases n-3 PUFA plasma concentrations and reduces AA.     

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.     

Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids

Long-chain unsaturated fatty acids, such as linoleic acid, show antibacterial activity and are the key ingredients of antimicrobial food additives and some antibacterial herbs. However, the precise mechanism for this antimicrobial activity remains unclear. We found that linoleic acid inhibited bacterial enoyl-acyl carrier protein reductase (FabI), an essential component of bacterial fatty acid synthesis, which has served as a promising target for antibacterial drugs. Additional unsaturated fatty acids including palmitoleic acid, oleic acid, linolenic acid, and arachidonic acid also exhibited the inhibition of FabI. However, neither the saturated form (stearic acid) nor the methyl ester of linoleic acid inhibited FabI. These FabI-inhibitory activities of various fatty acids and their derivatives very well correlated with the inhibition of fatty acid biosynthesis using [(14)C] acetate incorporation assay, and importantly, also correlated with antibacterial activity. Furthermore, the supplementation with exogenous fatty acids reversed the antibacterial effect of linoleic acid, which showing that it target fatty acid synthesis. Our data demonstrate for the first time that the antibacterial action of unsaturated fatty acids is mediated by the inhibition of fatty acid synthesis.     

Diversity of the fatty acids of the Nostoc species and their statistical analysis

Low molecular, hydroxy, dioic, saturated and unsaturated fatty acids were determined of six cyanobacterial species belonging to genus Nostoc and in different habitats: freshwater, terrestrial, and as well as symbionts. There are large variations in individual fatty acid contents according to species, and location of the genus Nostoc. Statistical analysis of variability of fatty acids belonging to the genus Nostoc is reported.     

Chemical C2-elongation of polyunsaturated fatty acids

Three fatty acids were synthesized from commercially available alpha-linolenic, stearidonic and eicosapentaenoic acids by C2-elongation using a four step preparative technique. The parent fatty acid methyl esters were reduced to alcohols with LiAlH(4), converted to bromides by treatment with triphenylphosphine dibromide, coupled with a lithiated C2-elongation block--2,4,4-trimethyl-2-oxazoline--to form the corresponding 2,2-dimethyloxazolines of C2-elongated fatty acids, and finally, converted to the target polyunsaturated fatty acids by acidic alcoholysis. Yields of more than 60% were achieved on a gram scale. The resulting 11Z,14Z,17Z-eicosatrienoic, 8Z,11Z,14Z,17Z-eicosatetraenoic and 7Z,10Z,13Z,16Z,19Z-docosapentaenoic acids were obtained as colorless oils with >98% purity and could be used for biochemical investigations without additional purification. The elongated fatty acids were free of byproducts that could result from Z-E isomerization or migration of double bonds.     

Estimation of the stoichiometry of volatile fatty acid production in the rumen of lactating cows

The purpose of this study was to improve the prediction of the quantity and type of Volatile Fatty Acids (VFA) produced from fermented substrate in the rumen of lactating cows. A model was formulated that describes the conversion of substrate (soluble carbohydrates, starch, hemi-cellulose, cellulose, and protein) into VFA (acetate, propionate, butyrate, and other VFA). Inputs to the model were observed rates of true rumen digestion of substrates, whereas outputs were observed molar proportions of VFA in rumen fluid. A literature survey generated data of 182 diets (96 roughage and 86 concentrate diets). Coefficient values that define the conversion of a specific substrate into VFA were estimated meta-analytically by regression of the model against observed VFA molar proportions using non-linear regression techniques. Coefficient estimates significantly differed for acetate and propionate production in particular, between different types of substrate and between roughage and concentrate diets. Deviations of fitted from observed VFA molar proportions could be attributed to random error for 100%. In addition to regression against observed data, simulation studies were performed to investigate the potential of the estimation method. Fitted coefficient estimates from simulated data sets appeared accurate, as well as fitted rates of VFA production, although the model accounted for only a small fraction (maximally 45%) of the variation in VFA molar proportions. The simulation results showed that the latter result was merely a consequence of the statistical analysis chosen and should not be interpreted as an indication of inaccuracy of coefficient estimates. Deviations between fitted and observed values corresponded to those obtained in simulations.     

Omega-3 fatty acids in neurodegenerative diseases: Focus on mitochondria

Mitochondrial dysfunction represents a common early pathological event in brain aging and in neurodegenerative diseases, e.g., in Alzheimer’s (AD), Parkinson’s (PD), and Huntington’s disease (HD), as well as in ischemic stroke. In vivo and ex vivo experiments using animal models of aging and AD, PD, and HD mainly showed improvement of mitochondrial function after treatment with polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA). Thereby, PUFA are particular beneficial in animals treated with mitochondria targeting toxins. However, DHA showed adverse effects in a transgenic PD mouse model and it is not clear if a diet high or low in PUFA might provide neuroprotective effects in PD. Post-treatment with PUFA revealed conflicting results in ischemic animal models, but intravenous administered DHA provided neuroprotective efficacy after acute occlusion of the middle cerebral artery. In summary, the majority of preclinical data indicate beneficial effects of n-3 PUFA in neurodegenerative diseases, whereas most controlled clinical trials did not meet the expectations. Because of the high half-life of DHA in the human brain clinical studies may have to be initiated much earlier and have to last much longer to be more efficacious.