Identification of cis-9,10-methylenehexadecanoic acid in submitochondrial particles of bovine heart

Submitochondrial particles of bovine heart were hydrolyzed by phospholipase A₂ and the products were analyzed by liquid chromatography electrospray ionization-mass spectrometry. We found a fatty acid with a molecular mass of 268 Da and a retention time longer than that of linoleic acid. Next, we synthesized organically cis-9,10-methylenehexadecanoic acid, which has a molecular mass similar to that of the extracted fatty acid, and characterized its high performance liquid chromatography and gas chromatography-mass spectrometry profiles. Using these data we were able to identify endogenous cis-9,10-methylenehexadecanoic acid in rat and human heart and liver tissues that had been hydrolyzed by phospholipase A₂. This fatty acid was not detected in tissue extracts that had not been hydrolyzed by phospholipase A₂. Similar amounts of cis-9,10-methylenehexadecanoic acid were measured in tissue extracts after total hydrolysis. These results suggest that cis-9,10-methylenehexadecanoic acid is a fatty acid component, in the sn-2 position, of phospholipids in some mammalian tissue.     

High myristic acid content in the cyanobacterium Cyanothece sp. PCC 8801 results from substrate specificity of lysophosphatidic acid acyltransferase

Analysis of fatty acids from the cyanobacterium Cyanothece sp. PCC 8801 revealed that this species contained high levels of myristic acid (14:0) and linoleic acid in its glycerolipids, with minor contributions from palmitic acid (16:0), stearic acid, and oleic acid. The level of 14:0 relative to total fatty acids reached nearly 50%. This 14:0 fatty acid was esterified primarily to the sn-2 position of the glycerol moiety of glycerolipids. This characteristic is unique because, in most of the cyanobacterial strains, the sn-2 position is esterified exclusively with C16 fatty acids, generally 16:0. Transformation of Synechocystis sp. PCC 6803 with the PCC8801_1274 gene for lysophosphatidic acid acyltransferase (1-acyl-sn-glycerol-3-phosphate acyltransferase) from Cyanothece sp. PCC 8801 increased the level of 14:0 from 2% to 17% in total lipids and the increase in the 14:0 content was observed in all lipid classes. These findings suggest that the high content of 14:0 in Cyanothece sp. PCC 8801 might be a result of the high specificity of this acyltransferase toward the 14:0-acyl-carrier protein.     

Origin of cyclohexanecarboxylic acid in Bacillus acidocaldarius

In Bacillus acidocaldarius, shikimic acid is converted into the cyclohexancearboxylic acid precursor of fatty acids by way of cyclohexene-l-carboxylic acid, but not by way of cyclohexene-3- or -4-carboxylic acid or benzoic acid.     

Generation of fad2 transgenic mice that produce omega-6 fatty acids

Fatty acid desaturase-2 (FAD2) introduces a double bond in position Δ12 in oleic acid (18︰1) to form linoleic acid (18︰2 n-6) in higher plants and microbes. A new transgenic expression cassette, containing CMV promoter/fad2 cDNA/SV40 polyA, was constructedto produce transgenic mice. Among 63 healthy offspring, 10 founders (15.9%) integrated the cotton fad2 transgene into their genomes, as demonstrated by PCR and Southern blotting analysis. All founder mice were fertile and heterozygous fad2 female and nontransgenic littermates were used for fatty acid analysis using gas chromatography. One fad2 transgenic line showed substantial differences in the fatty acid profiles and the level of linoleic acid was increased 19% (P<0.05) in transgenic muscles compared to their nontransgenic littermates. Moreover, it exhibited an 87% and a 9% increase (P<0.05) in arachidonic acid (20︰4 n-6) in muscles and liver, compared to their nontransgenic littermates. The results indicate that the plant fad2 gene can be functionally expressed in transgenic mice and may playan active role in conversion of oleic acid into linoleic acid.     

Characterization of cis-9 trans-11 trans-15 C18:3 in milk fat by GC and covalent adduct chemical ionization tandem MS

Rumen biohydrogenation of dietary α-linolenic acid gives rise in ruminants to accumulation of fatty acid intermediates, some of which may be transferred into milk. Rumelenic acid [cis-9 trans-11 cis-15 C18:3 (RLnA)] has recently been characterized, but other C18:3 minor isomers are still unknown. The objective of this work was to identify a new isomer of octatridecenoic acid present in milk fat from ewes fed different sources of α-linolenic acid. Structural characterization of this fatty acid was achieved by GC-MS. Analysis of dimethyloxazoline and picolinyl ester derivatives allowed for location of the double bond positions. Covalent adduct chemical ionization tandem mass spectrometry confirmed the positional structure 9-11-15, identical to RLnA, and helped to establish double bond geometry (cis-trans-trans). This new C18:3 isomer could be formed by isomerization of cis-15 bond of RLnA and subsequently converted by hydrogenation to trans-11 trans-15 C18:2, an octadecadienoic acid also detected in this study.     

Lactic acid bacteria isolated from fish gut produce conjugated linoleic acid without the addition of exogenous substrate

The production of conjugated linoleic acid (CLA) by four strains of lactic acid bacteria isolated from fish, i.e., Leuconostoc mesenteroides H20, Leuconostoc mesenteroides H22, Leuconostoc lactis H24 and Lactobacillus pentosus H16, was evaluated in MRS broth and on MRS agar. The bioconversion and production of CLA by resting cells were also assessed. Linoleic acid was detected in cultures grown on agar at percentages of up to 18.3% (w/w) of total fatty acid, and conjugated isomers were found in the fatty acid profiles of Lactobacillus pentosus H16. The percentage of CLA relative to total fatty acid increased from 5.68 ± 1.65% to 23.69 ± 0.79% when resting cells were removed from agar plates and incubated without the addition of exogenous linoleic acid as a substrate. When Lactobacillus pentosus H16 cells were incubated with linoleic acid, cyclization and changes in monounsaturated fatty acid percentages were observed instead of conjugation. These results show that growth on a solid support is required for CLA production. More significantly, an increase in the CLA content could be achieved by incubating resting cells without exogenous substrate.     

A role for the human peroxisomal half-transporter ABCD3 in the oxidation of dicarboxylic acids

Peroxisomes play a major role in human cellular lipid metabolism, including fatty acid β-oxidation. Free fatty acids (FFAs) can enter peroxisomes through passive diffusion or by means of ATP binding cassette (ABC) transporters, including HsABCD1 (ALDP, adrenoleukodystrophy protein), HsABCD2 (ALDRP) and HsABCD3 (PMP70). The physiological functions of the different peroxisomal half-ABCD transporters have not been fully determined yet, but there are clear indications that both HsABCD1 and HsABCD2 are required for the breakdown of fatty acids in peroxisomes. Here we report that the phenotype of the pxa1/pxa2Δ yeast mutant, i.e. impaired oxidation of oleic acid, cannot only be partially rescued by HsABCD1, HsABCD2, but also by HsABCD3, which indicates that each peroxisomal half-transporter can function as homodimer. Fatty acid oxidation measurements using various fatty acids revealed that although the substrate specificities of HsABCD1, HsABCD2 and HsABCD3 are overlapping, they have distinctive preferences. Indeed, most hydrophobic C24:0 and C26:0 fatty acids are preferentially transported by HsABCD1, C22:0 and C22:6 by HsABCD2 and most hydrophilic substrates like long-chain unsaturated-, long branched-chain- and long-chain dicarboxylic fatty acids by HsABCD3. All these fatty acids are most likely transported as CoA esters. We postulate a role for human ABCD3 in the oxidation of dicarboxylic acids and a role in buffering fatty acids that are overflowing from the mitochondrial β-oxidation system.     

Acyltransferases and transacylases that determine the fatty acid composition of glycerolipids and the metabolism of bioactive lipid mediators in mammalian cells and model organisms

Over one hundred different phospholipid molecular species are known to be present in mammalian cells and tissues. Fatty acid remodeling systems for phospholipids including acyl-CoA:lysophospholipid acyltransferases, CoA-dependent and CoA-independent transacylation systems, are involved in the biosynthesis of these molecular species. Acyl-CoA:lysophospholipid acyltransferase system is involved in the synthesis of phospholipid molecular species containing sn-1 saturated and sn-2 unsaturated fatty acids. The CoA-dependent transacylation system catalyzes the transfer of fatty acids esterified in phospholipids to lysophospholipids in the presence of CoA without the generation of free fatty acids. The CoA-dependent transacylation reaction in the rat liver exhibits strict fatty acid specificity, i.e., three types of fatty acids (20:4, 18:2 and 18:0) are transferred. On the other hand, CoA-independent transacylase catalyzes the transfer of C20 and C22 polyunsaturated fatty acids from diacyl phospholipids to various lysophospholipids, especially ether-containing lysophospholipids, in the absence of any cofactors. CoA-independent transacylase is assumed to be involved in the accumulation of PUFA in ether-containing phospholipids. These enzymes are involved in not only the remodeling of fatty acids, but also the synthesis and degradation of some bioactive lipids and their precursors. In this review, recent progresses in acyltransferase research including the identification of the enzyme’s genes are described.     

Fatty acid composition of Mesorhizobium huakuii lipopolysaccharides. Identification of 27-oxooctacosanoic acid

Lipopolysaccharides of three Mesorhizobium huakuii strains carried a number of amide-linked 3-hydroxylated fatty acids including: 3-OH-12:0, 3-OH-i-13:0, 3-OH-20:0, 3-OH-i-21:0, 3-OH-22:0, 3-OH-23:0 and unsaturated 3-OH-22:1. The first three of the above mentioned acids are the main amide-linked fatty acids in the LPS preparations. The main ester-bound fatty acids comprise 16:0, i-17:0, 18:0, 20:0 and 27-OH-28:0. Among minor constituents of lipid A 25-OH-26:0 and 29-OH-30:0 together with some non-polar fatty acids were found. Additionally, the presence of 4-oxo-20:0, 4-oxo-i-21:0 and 4-oxo-22:0 amide-bound fatty acids as well as the 27-oxo-28:0 ester-linked fatty acid were proved. To our knowledge oxo fatty acids are rare constituents of lipopolysaccharides and 27-oxo-28:0 was found for the first time in the LPS preparations from members of Rhizobiaceae.     

Alteration of the omega-3 fatty acid desaturase gene is associated with reduced linolenic acid in the A5 soybean genotype

Reducing the linolenic acid (18?:?3^{ω? 3,6,9}) concentration of soybean [Glycine max (L.) Merr.] oil may lessen the need for chemical hydrogenation and enhance flavor stability. Soybean genotypes A5 and A23 have reduced linolenic acid concentration compared with current cultivars. Seed linolenic acid is synthesized primarily by the ω-3 fatty acid desaturase located in the microsomes. The objective of this research was to study whether this enzyme has a role in reducing the fatty acid levels in the soybean genotypes A5 and A23. DNA from A5 and A23 was analyzed by gel-blot hybridization with a cDNA encoding the ω-3 fatty acid desaturase. A5 and lines selected from it have a DNA fragment missing compared to A23 and lines with normal linolenic acid concentration. Seventy F_4:5 lines from a population segregating for linolenic acid concentration were scored for presence or absence of the fragment. The absence of the fragment was significantly (P?0.0001) associated with a reduced linolenic acid level and accounted for 67% of the variation for linolenic acid in the population. These results suggest that the reduced linolenic acid concentration in A5 was at least partially the result of a full or partial deletion of a microsomal ω-3 desaturase gene. No DNA polymorphisms were found for the desaturase gene in A23, so no mutations could be studied in this line.     

Overexpression of stearoyl-CoA desaturase-1 results in an increase of conjugated linoleic acid (CLA) and n-7 fatty acids in 293 cells

The effect of human SCD1 heterologous expression on cellular fatty acid synthesis was investigated in the current study. The SCD1 gene expression cassette and PGK-neomycin-selectable marker cassette were co-introduced into HEK 293 cells by electroporation, and subsequently, SCD1 expression was evaluated by fatty acid analysis. RT-PCR analysis indicated that the foreign SCD1 gene could be expressed in transformed cell lines. Total lipid analysis of the transformed cells fed with vaccenic acid (t11-18:1) as a substrate showed that SCD1 expression resulted in an increase in c9t11-CLA from 0.73-1.03% to 2.69-2.86% (p < 0.05) and that the conversion efficiency was elevated from 5.11-6.88% to 16.49-20.06% (p < 0.05). Surprisingly, the concentration of t10c12-CLA was also increased, from 0.10-0.41% to 1.35-1.69% in SCD1 cells (p < 0.05). SCD1 expression also resulted in a significant (p < 0.05) increase in palmitoleic acid (16:1 n-7) from 1.56-2.26% to 3.47-4.04% and cis-vaccenic acid (18:1 n-7) from 2.42-3.97% to 6.20-7.22%, and the corresponding conversion ratio of n-7 fatty acid was elevated from 12.01-16.70% to 22.62-24.13% (p < 0.05). This study demonstrates that the foreign SCD1 gene was expressed with high efficiency and induced elevated c9t11-CLA, t10c12-CLA, and n-7 fatty acid levels in mammalian cells.     

Medium-chain fatty acids enhance expression and histone acetylation of genes related to lipid metabolism in insulin-resistant adipocytes

BackgroundThe expressions of genes related to lipid metabolism are decreased in adipocytes with insulin resistance. In this study, we examined the effects of fatty acids on the reduced expressions and histone acetylation of lipid metabolism-related genes in 3T3-L1 adipocytes treated with insulin resistance induced by tumor necrosis factor (TNF)-α.MethodsShort-, medium-, and long-chain fatty acid were co-administered with TNF-α in 3T3-L1 adipocytes. Then, mRNA expressions and histone acetylation of genes involved in lipid metabolism were determined using mRNA microarrays, qRT-PCR, and chromatin immunoprecipitation assays.ResultsWe found in microarray and subsequent qRT-PCR analyses that the expression levels of several lipid metabolism-related genes, including Gpd1, Cidec, and Cyp4b1, were reduced by TNF-α treatment and restored by co-treatment with a short-chain fatty acid (C4: butyric acid) and medium-chain fatty acids (C8: caprylic acid and C10: capric acid). The pathway analysis of the microarray showed that capric acid enhanced mRNA levels of genes in the PPAR signaling pathway and adipogenesis genes in the TNF-α-treated adipocytes. Histone acetylation around Cidec and Gpd1 genes were also reduced by TNF-α treatment and recovered by co-administration with short- and medium-chain fatty acids.General significanceMedium- and short-chain fatty acids induce the expressions of Cidec and Gpd1, which are lipid metabolism-related genes in insulin-resistant adipocytes, by promoting histone acetylation around these genes.     

Partial purification and characterization of the lipoxygenase from grains of Hordeum distichum

Lipoxygenase activities in ungerminated and germinating barley grains were found to be associated exclusively with the embryos. A lipoxygenase was extracted from ungerminated embryos and partially purified by fractional precipitation with ammonium sulfate and gel-filtration. Both the crude extracts and the purified preparation appeared to contain only a fatty acid type lipoxygenase which mainly converted linolele acid to 9-hydroperoxy, trans-10, cis-12-octadecadienoic acid. The purified enzyme was inhibited by its own products, hydroperoxides, but not by 1 mM cyanide, EDTA, Hg²⁺ or Cu²⁺.     

Lipoxygenase-mediated cleavage of fatty acids to carbonyl fragments in tomato fruits

Homogenates of tomato fruits catalysed the enzymic conversion of linoleic and linolenic acids (but not oleic acid) to C₆ aldehydes in low (3–5%) molar yield. Hexanal was formed from linoleic acid; cis-3-hexenal and smaller amounts of trans-2-hexenal were formed from linolenic acid. With the fatty acids as substrates, the major products were fatty acid hydroperoxides (50–80% yield) and the ratio of 9- to 13-hydroperoxides as isolated from an incubation with linoleic acid was at least 95:5 in favour of the 9-hydroperoxide isomer. When the 9- and 13-hydroperoxides of linoleic acid were used as substrates with tomato homogenates, the 13-hydroperoxide was readily cleaved to hexanal in high molar yield (60%) but the 9-hydroperoxide isomer was not converted to cleavage products. Properties of the hydroperoxide cleavage system are described. The results indicate that the C₆ aldehydes are formed from C₁₈ polyunsaturated fatty acids in a sequential enzyme system involving lipoxygenase (which preferentially oxygenates at the 9-position) followed by a hydroperoxide cleavage system which is, however, specific for the 13-hydroperoxy isomers.     

Analysis of the promoter region of the gene LIP1 encoding triglyceride lipase from Fusarium graminearum

Triglyceride lipases catalyze the reversible degradation of glycerol esters with long-chain fatty acids into fatty acids and glycerol. In silico analysis of 5′-end flanking sequence of the gene LIP1 encoding a triglyceride lipase from the wheat head blight pathogen Fusarium graminearum revealed the presence of several cis-regulatory elements. To delineate the function of these regulatory elements, we constructed a series of deletion mutants in the LIP1 promoter region fused to the open reading frame of a green fluorescent protein (GFP) and assayed the promoter activity. Analysis of GFP expression levels in mutants indicated that a 563-bp promoter sequence was sufficient to drive the expression of LIP1 and regulatory elements responsible for the gene induction were located within the 563-372 bp region. To further investigate the regulatory elements, putative cis-acting elements spanned within the 563-372 bp region were mutated using a targeted mutagenesis approach. A CCAAT box, a CreA binding site, and a fatty acid responsive element (FARE) were identified and confirmed to be required for the basal expression of LIP1, glucose suppression and fatty acid induction, respectively.     

Flavour biogenesis. Partial purification and properties of a fatty acid hydroperoxide cleaving enzyme from fruits of cucumber

A membrane-bound enzyme, which catalyses the cleavage of fatty acid hydroperoxides to carbonyl fragments, has been partially purified from cucumber fruit. The isomeric 9- and 13-hydroperoxydienes (but not the hydroxydienes) derived from both linoleic and linolenic acids are cleaved by the enzyme but a mixture of 9- and 10-hydroperoxymonoenoic derivatives of oleic acid was not attacked. No evidence was obtained for free intermediates between fatty acid hydroperoxides and the cleavage products. Major volatile products were: cis-3-nonenal and hexanal (from 9- and 13-hydroperoxides of linoleic acid respectively) or cis-3,cis-6-nonadienal and cis-3-hexenal (from 9- and 13-hydroperoxides of linolenic acid). The increase in the ratio of cis-3- to trans-2-enal products with enzyme purification indicated that cis-3-enals are the immediate cleavage products and that the trans-2- forms are produced by subsequent isomerization.     

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.     

Biochemical characterization and FAD-binding analysis of oleate hydratase from Macrococcus caseolyticus

A putative fatty acid hydratase gene from Macrococcus caseolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was a 68 kDa dimer with a molecular mass of 136 kDa. The enzymatic products formed from fatty acid substrates by the putative enzyme were isolated with high purity (>99%) by solvent fractional crystallization at low temperature. After the identification by GC–MS, the purified hydroxy fatty acids were used as standards to quantitatively determine specific activities and kinetic parameters for fatty acids as substrates. Among the fatty acids evaluated, specific activity and catalytic efficiency (k_cat/K_m) were highest for oleic acid, indicating that the putative fatty acid hydratase was an oleate hydratase. Hydration occurred only for cis-9-double and cis-12-double bonds of unsaturated fatty acids without any trans-configurations. The maximum activity for oleate hydration was observed at pH 6.5 and 25 °C with 2% (v/v) ethanol and 0.2 mM FAD. Without FAD, all catalytic activity was abolished. Thus, the oleate hydratase is an FAD-dependent enzyme. The residues G29, G31, S34, E50, and E56, which are conserved in the FAD-binding motif of fatty acid hydratases (GXGXXG_(A/S)X_(15–21)E_(D)), were selected by alignment, and the spectral properties and kinetic parameters of their alanine-substituted variants were analyzed. Among the five variants, G29A, G31A, and E56A showed no interaction with FAD and exhibited no activity. These results indicate that G29, G31, and E56 are essential for FAD-binding.