Catabolism of (2E)-4-Hydroxy-2-nonenal via ω- and ω-1-Oxidation Stimulated by Ketogenic Diet

Oxidative stress triggers the peroxidation of ω-6-polyunsaturated fatty acids to reactive lipid fragments, including (2E)-4-hydroxy-2-nonenal (HNE). We previously reported two parallel catabolic pathways of HNE. In this study, we report a novel metabolite that accumulates in rat liver perfused with HNE or 4-hydroxynonanoic acid (HNA), identified as 3-(5-oxotetrahydro-2-furanyl)propanoyl-CoA. In experiments using a combination of isotopic analysis and metabolomics studies, three catabolic pathways of HNE were delineated following HNE conversion to HNA. (i) HNA is ω-hydroxylated to 4,9-dihydroxynonanoic acid, which is subsequently oxidized to 4-hydroxynonanedioic acid. This is followed by the degradation of 4-hydroxynonanedioic acid via β-oxidation originating from C-9 of HNA breaking down to 4-hydroxynonanedioyl-CoA, 4-hydroxyheptanedioyl-CoA, or its lactone, 2-hydroxyglutaryl-CoA, and 2-ketoglutaric acid entering the citric acid cycle. (ii) ω-1-hydroxylation of HNA leads to 4,8-dihydroxynonanoic acid (4,8-DHNA), which is subsequently catabolized via two parallel pathways we previously reported. In catabolic pathway A, 4,8-DHNA is catabolized to 4-phospho-8-hydroxynonanoyl-CoA, 3,8-dihydroxynonanoyl-CoA, 6-hydroxyheptanoyl-CoA, 4-hydroxypentanoyl-CoA, propionyl-CoA, and acetyl-CoA. (iii) The catabolic pathway B of 4,8-DHNA leads to 2,6-dihydroxyheptanoyl-CoA, 5-hydroxyhexanoyl-CoA, 3-hydroxybutyryl-CoA, and acetyl-CoA. Both in vivo and in vitro experiments showed that HNE can be catabolically disposed via ω- and ω-1-oxidation in rat liver and kidney, with little activity in brain and heart. Dietary experiments showed that ω- and ω-1-hydroxylation of HNA in rat liver were dramatically up-regulated by a ketogenic diet, which lowered HNE basal level. HET0016 inhibition and mRNA expression level suggested that the cytochrome P450 4A are main enzymes responsible for the NADPH-dependent ω- and ω-1-hydroxylation of HNA/HNE.     

Association of the FADS2 Gene with ω-6 and ω-3 PUFA Concentration in the Egg Yolk of Japanese Quail

This study focused on the association of polymorphisms of the FADS2 gene with fatty acid profiles in egg yolk of eight Japanese quail lines selected for high and low ω-6:ω-3 PUFA ratio (h2 = 0.36–0.38). For the identification of polymorphisms within the FADS2 gene 1350 bp of cDNA sequence were obtained encoding 404 amino acids. Five synonymous SNPs were found by comparative sequencing of animals of the high and low lines. These SNPs were genotyped by single base extension on 160 Japanese quail. The association analysis, comprising analysis of variance and family based association test (FBAT), revealed significant effects of SNP3 and SNP4 genotypes on the egg yolk fatty acid profiles, especially the ω-6 and ω-3 PUFAs (P < 0.05). No effects of the other SNPs were found—indicating that these are not in linkage disequilibrium with the causal polymorphism. The results of this study promote FADS2 as a functional candidate gene for traits related to ω-6 and ω-3 PUFA concentration in the egg yolk.     

Influence of Exogenous Natural Oils on the ω-1 and ω-2 Hydroxy Fatty Acid Moiety of Sophorose Lipid Produced by Candida bombicola

Candida bombicola can synthesize monohydroxy fatty acid as a moiety of sophorose lipids. The hydroxy fatty acids contained in a major lactone were identified by GC-MS, after culturing with natural oils such as coconut, rapeseed, olive, and soybean oils. Hydroxy fatty acids of C18 and C16 were always synthesized, but differences were observed among the oils regarding the positions of hydroxyl groups, unsaturation, and composition of the fatty acids. A new C17 hydroxy acid was found without addition of oil.     

Δ6-Desaturase (FADS2) deficiency unveils the role of ω3- and ω6-polyunsaturated fatty acids

Mammalian cell viability is dependent on the supply of the essential fatty acids (EFAs) linoleic and α-linolenic acid. EFAs are converted into ω3- and ω6-polyunsaturated fatty acids (PUFAs), which are essential constituents of membrane phospholipids and precursors of eicosanoids, anandamide and docosanoids. Whether EFAs, PUFAs and eicosanoids are essential for cell viability has remained elusive. Here, we show that deletion of Δ6-fatty acid desaturase (FADS2) gene expression in the mouse abolishes the initial step in the enzymatic cascade of PUFA synthesis. The lack of PUFAs and eicosanoids does not impair the normal viability and lifespan of male and female fads2−/− mice, but causes sterility. We further provide the molecular evidence for a pivotal role of PUFA-substituted membrane phospholipids in Sertoli cell polarity and blood–testis barrier, and the gap junction network between granulosa cells of ovarian follicles. The fads2−/− mouse is an auxotrophic mutant. It is anticipated that FADS2 will become a major focus in membrane, haemostasis, inflammation and atherosclerosis research.     

Impact of ω-5 gliadin on wheat-dependent exercise-induced anaphylaxis in mice

The effects of ω-5 gliadin on wheat-dependent exercise-induced anaphylaxis (WDEIA) were investigated by using a mouse model. The gliadin fraction was prepared as a 70% ethanol-soluble solution, and ω-5 gliadin was purified by chromatography. Purified ω-5 gliadin was run on SDS-PAGE gel to reveal three bands with a molecular mass of 53-60 kDa and had the characteristic N-terminal sequence of ω-5 gliadin. The mice were sensitized to the gliadin fraction, and the anaphylactic response was assessed by measuring the body temperature and voluntary physical activity. An oral administration of ω-5 gliadin evoked a significant drop in both the body temperature and voluntary physical activity, similar to the effects of the whole gliadin fraction. ELISA and immunoblotting analyses revealed that the IgE expression from sensitized mice reacted most strongly to ω-5 gliadin. Taken together, these results indicate ω-5 gliadin to be a major allergen responsible for stimulating WDEIA in mice, with the characteristic potential for stimulating IgE production.     

Diverse rare lipid-related metabolites including ω-7 and ω-9 alkenylitaconic acids (ceriporic acids) secreted by a selective white rot fungus, Ceriporiopsis subvermispora

Ceriporiopsis subvermispora is a selective white-rot fungus that secretes alk(en)ylitaconic acids named ceriporic acids, known as ion redox silencers. In this study, we analysed a series of extracellular lipid-related metabolites produced by the fungus and found that a wide variety of ceriporic acids and fatty acids, including those with odd-numbered and very long-chains, were produced in wood meal cultures. Two new ceriporic acids, (R)-3-[(Z)-tetradec-7-enyl]-itaconic acid (ceriporic acid E) and (R)-3-[(Z)-tetradec-5-enyl]-itaconic acid (ceriporic acid F), were for the first time identified by dimethyl disulfide derivatisation, followed by GC/EI-MS, (1)H and homonuclear J-resolved 2D NMR and feeding experiments with [(13)C-U] glucose coupled with multiple-stage mass spectrometry. In separation by GC and LC, a reversed correlation of elution sequences between a nonpolar GC column and an ODS-LC column for cis and trans isomers of ω7 and ω9 lipids was found, and the elution of new metabolites was in accordance with the prevailing theory. The biosynthetic precursors of ceriporic acid F can be proposed as oxaloacetate and 16:1Δ7-CoA. Because fatty acids biosynthesised from 16:1Δ7-CoA have been reported for only a limited number of organisms, the highly individual structure of ceriporic acid F is highlighted.     

Heterologous expression of Brassica juncea microsomal ω-3 desaturase gene (BjFad3) improves the nutritionally desirable ω-6:ω-3 fatty acid ratio in rice bran oil

Rice bran oil (RBO), being naturally rich in antioxidants, is currently regarded as one of the health-beneficial edible oils. However, the RBO has essential linoleic acid (ω-6, C18:2) and α-linolenic acid (ω-3, C18:3) in nutritionally disproportionate level (~25:1), contrary to the WHO/FAO’s recommendation of ~5:1. Among few naturally occurring C18:3 enriched oil-seeds, Brassica juncea (Indian mustard) has almost equal proportion of ω-6 and ω-3 fatty acids in its oil due to the activity of microsomal ω-3 desaturase (Fad3), which converts C18:2–C18:3. Therefore, the full length Fad3 coding DNA sequence (CDS) was isolated from the developing seeds of B. juncea, functionally characterized and heterologously expressed for the nutritional enhancement of RBO. Sequence analysis revealed that the 1,134 bp long BjFad3 CDS corresponds to a polypeptide of 377 amino acids, which is highly (85–95 %) homologous to other known Fad3 enzymes of plant kingdom. The BjFad3 gene was initially characterized in transgenic tobacco to establish its linoleate desaturase activity. Thereafter, rice bran-specific expression of the BjFad3 was carried out to alter the fatty acid profile of RBO. Several independent transgenic lines of tobacco and rice plants were developed by Agrobacterium-mediated transformation. Standard molecular biological techniques were used to confirm the transgene integration in the respective genomes and subsequent in planta expression. The BjFad3 transgene expression correlated to the significant increase in C18:3 fatty acid content (up to tenfold) in both tobacco seed oil and RBO, and thereby improving the nutritionally desirable ω-6:ω-3 ratio (~2:1) in one of the transgenic rice lines.     

A general synthesis of long chain ω- and (ω-1)-hydroxy fatty acids

A method for the synthesis of long chain fatty acids substituted at the ω and ω-1 positions has been developed. The key step is the isomerization of the triple bond of an alkyn-1-ol from an internal position in the chain to the free terminus with a new, convenient reagent, sodium aminopropylamide (NaAPA). Standard functional group manipulations i.e., Jones oxidation, esterification and hydroboration of the triple bond are used to prepare ω-hydroxy fatty esters. The generality of the method is illustrated with syntheses of ω-hydroxy fatty esters with 24, 26, 28 and 30 carbon chains.In the 24 carbon series, hydration of the terminal triple bond of alkynoic ester 4a followed by reduction gave the (ω-1)-hydroxy ester.     

ω-ethyl, ω-ethenyl and ω-ethynyl-α-alkylidene-γ-lactones from Clinostemon mahuba

The trunk wood of Clinostemon mahuba contains eight (3R)-2-alkylidene-3-hydroxy-4-methylenebutanolides, seven (3R,4S)-2-alkylidene-3-hydroxy-4-methylbutanolides and seven (3S,4S)-2-alkylidene-3-hydroxy-4-methylbutanolides distinguished by the alkylidene side chains with respect to their E- or Z-geometry, ethenyl, ethynyl or ethyl terminals and lengths (C₁₆ or C₁₈).     

One Single Basic Amino Acid at the ω-1 or ω-2 Site Is a Signal That Retains Glycosylphosphatidylinositol-Anchored Protein in the Plasma Membrane of Aspergillus fumigatus

Although the plasma membrane is the terminal destination for glycosylphosphatidylinositol (GPI) proteins in higher eukaryotes, cell wall-attached GPI proteins (GPI-CWPs) are found in many fungal species. In yeast, some of the cis-requirements directing localization of GPI proteins to the plasma membrane or cell wall are now understood. However, it remains to be determined how Aspergillus fumigatus, an opportunistic fungal pathogen, signals, and sorts GPI proteins to either the plasma membrane or the cell wall. In this study, chimeric green fluorescent proteins (GFPs) were constructed as fusions with putative C-terminal GPI signal sequences from A. fumigatus Mp1p, Gel1p, and Ecm33p, as well as site-directed mutations thereof. By analyzing cellular localization of chimeric GFPs using Western blotting, electron microscopy, and fluorescence microscopy, we showed that, in contrast to yeast, a single Lys residue at the ω-1 or ω-2 site alone could retain GPI-anchored GFP in the plasma membrane. Although the signal for cell wall distribution has not been identified yet, it appeared that the threonine/serine-rich region at the C-terminal half of AfMp1 was not required for cell wall distribution. Based on our results, the cis-requirements directing localization of GPI proteins in A. fumigatus are different from those in yeast.     

Biotechnological production and applications of the ω-3 polyunsaturated fatty acid docosahexaenoic acid

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid composed of 22 carbon atoms and six double bonds. Because the first double bond, as counted from the methyl terminus, is at position three, DHA belongs to the so-called -3 group. In recent years, DHA has attracted much attention because of its beneficial effect on human health. At present, fish oil is the major source of DHA, but alternatively it may be produced by use of microorganisms. Marine microorganisms may contain large quantities of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light. These processes can be well controlled and DHA with constant quality can be produced all year round. This paper reviews recent advances in the biotechnological production of DHA by marine microorganisms.     

Arachidonic Acid-metabolizing Cytochrome P450 Enzymes Are Targets of ω-3 Fatty Acids

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J isoforms converting AA to epoxyeicosatrienoic acids (EETs) preferentially epoxidized the ω-3 double bond and thereby produced 17,18-epoxyeicosatetraenoic (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) from EPA and DHA. We found that these ω-3 epoxides are highly active as antiarrhythmic agents, suppressing the Ca²⁺-induced increased rate of spontaneous beating of neonatal rat cardiomyocytes, at low nanomolar concentrations. CYP4A/4F isoforms ω-hydroxylating AA were less regioselective toward EPA and DHA, catalyzing predominantly ω- and ω minus 1 hydroxylation. Rats given dietary EPA/DHA supplementation exhibited substantial replacement of AA by EPA and DHA in membrane phospholipids in plasma, heart, kidney, liver, lung, and pancreas, with less pronounced changes in the brain. The changes in fatty acids were accompanied by concomitant changes in endogenous CYP metabolite profiles (e.g. altering the EET/EEQ/EDP ratio from 87:0:13 to 27:18:55 in the heart). These results demonstrate that CYP enzymes efficiently convert EPA and DHA to novel epoxy and hydroxy metabolites that could mediate some of the beneficial cardiovascular effects of dietary ω-3 fatty acids.     

Grevillea robusta seed oil: A source of ω-5 monoenes

The fatty acids from Grevillea robusta seed oil triglycerides contain 22.5 % ω-5 monoenes ranging in chain length from C₁₄ to C₂₈. C₁₆ to C₂₆ saturates (18 %), C₁₈ to C₂₄ ω-9 monoenes (55 %), C₁₈ diene (2.3 %) and C₁₈ triene (0.7 %) make up the remainder of the acids.     

Substrate specificity and membrane topologies of the iron-containing ω3 and ω6 desaturases from Mortierella alpina

Applied Microbiology and Biotechnology - Polyunsaturated fatty acids (PUFAs) are essential lipids for cell function, normal growth, and development, serving as key structural components of...     

Features and technical applications of ω-transaminases

Chiral amines in enantiopure forms are important chemical building blocks, which are most well recognized in the pharmaceutical industries for imparting desirable biological activity to chemical entities. A number of synthetic strategies to produce chiral amines via biocatalytic as well as chemical transformation have been developed. Recently, ω-transaminase (ω-TA) has attracted growing attention as a promising catalyst which provides an environment-friendly access to production of chiral amines with exquisite stereoselectivity and excellent catalytic turnover. To obtain enantiopure amines using ω-TAs, either kinetic resolution of racemic amines or asymmetric amination of achiral ketones is employed. The latter is usually preferred because of twofold higher yield and no requirement of conversion of a ketone product back to racemic amine. However, the choice of a production process depends on several factors such as reaction equilibrium, substrate reactivity, enzyme inhibition, and commercial availability of substrates. This review summarizes the biochemical features of ω-TA, including reaction chemistry, substrate specificity, and active site structure, and then introduces recent advances in expanding the scope of ω-TA reaction by protein engineering and public database searching. We also address crucial factors to be considered for the development of efficient ω-TA processes.     

High throughput screening methods for ω-transaminases

Recently, ω-transaminases have been increasingly used to synthesize amine compounds by reductive amination of prochiral ketones which are of high pharmacological significance. However, the conventional methods for evaluating these enzymes are time consuming and have often been regarded as a bottle neck in developing these enzymes as industrial biocatalysts. In the past few years, several high throughput screening methods have been developed for fast evaluation and identification of ω-transaminase. This review summarizes the various methodologies developed for rapidly screening ω-transaminases.     

Partial rescue of Rett syndrome by ω-3 polyunsaturated fatty acids (PUFAs) oil

Evidence of enhanced oxidative stress (O.S.) and lipid peroxidation has been reported in patients with Rett syndrome (RTT), a relatively rare neurodevelopmental disorder progressing in 4-stages, and mainly caused by loss-of-function mutations in the methyl-CpG-binding protein 2. No effective therapy for preventing or arresting the neurologic regression in the disease in its various clinical presentations is available. Based on our prior evidence of enhanced O.S. and lipid peroxidation in RTT patients, herein we tested the possible therapeutic effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs), known antioxidants with multiple effects, on the clinical symptoms and O.S. biomarkers in the earliest stage of RTT. A total of 20 patients in stage I were randomized (n = 10 subjects per arm) to either oral supplementation with ω-3 PUFAs-containing fish oil (DHA: 72.9 ± 8.1 mg/kg b.w./day; EPA: 117.1 ± 13.1 mg/kg b.w./day; total ω-3 PUFAs: 246.0 ± 27.5 mg/kg b.w./day) for 6 months or no treatment. Primary outcomes were potential changes in clinical symptoms, with secondary outcomes including variations for five O.S. markers in plasma and/or erythrocytes (nonprotein bound iron, F₂-dihomo-isoprostanes, F₃-isoprostanes, F₄-neuroprostanes, and F₂-isoprostanes). A significant reduction in the clinical severity (in particular, motor-related signs, nonverbal communication deficits, and breathing abnormalities) together with a significant decrease in all the examined O.S. markers was observed in the ω-3 PUFAs supplemented patients, whereas no significant changes were evidenced in the untreated group. For the first time, these findings strongly suggest that a dietary intervention in this genetic disease at an early stage of its natural history can lead to a partial clinical and biochemical rescue.

Electronic supplementary material

The online version of this article (doi:10.1007/s12263-012-0285-7) contains supplementary material, which is available to authorized users.     

Improve freezing tolerance in transgenic poplar by overexpressing a ω-3 fatty acid desaturase gene

Polyunsaturated fatty acids are a major component of glycerolipids in plant cellular membranes, and play important role in the maintenance of its fluidity. In this study, we isolated a novel ω-3 fatty acid desaturase gene (PtFAD3) from Populus tomentosa Carr. and transformed it into hybrid poplar (Populus alba × P. glandulossa) K84 lines by overexpression and RNAi silencing methods. Quantitative PCR analysis revealed varied expression levels of PtFAD3 among the transgenic lines. Two overexpressing lines (OE lines) and two gene down-regulated RNAi lines (DR lines) were chosen for further analysis. The level of the major trienoic fatty acid species, linolenic acid, increased by an average of 10% in the two OE lines and decreased by an average of 5% in the two DR lines. When young poplar cuttings with three leaves were exposed to a freezing temperature (?4°C) for 3 h without cold acclimation, survival rates of the two independent OE lines (70 and 77%) were far superior to the wild type (36.7%) and the two DR lines (10% for both). Damage caused by freezing was alleviated significantly in the two OE lines compared to wild-type controls. Phenotypic evaluation indicated that the variable responses to freezing were attributable to genotypic variation. Therefore, in the case of poplar, a significant increase in freezing tolerance was achieved on a small scale following the introduction of PtFAD3. This strategy can be used for freezing tolerance breeding in hybrid poplar and other economically important forest trees.