Fatty acid synthesis from [2-14C]acetate in normal and peroxisome-deficient (Zellweger) fibroblasts

The incorporation of [2-¹⁴C]acetate into the lipids of normal and peroxisome-deficient (Zellweger's syndrome) skin fibroblasts was examined. Most of the label was incorporated into triacyglycerol fatty acids in normal as well as Zellweger's syndrome cells. Triacylglycerols and cholesteryl esters in Zellweger's syndrome cells contained increased levels of labelled saturated and monounsaturated very long-chain fatty acids (VLCFA, that is fatty acids with more than 22 carbon atoms), in particular hexacosanoic (26∶0) and hexacosaenoic (26∶1) acids. As traces of labelled VLCFA with up to 32 carbon atoms were detected in triacylglycerols even in control cells it is probable that these fatty acids are formed naturally during the elongation process. Our data suggest that peroxisomes are involved in the chain shortening of the saturated and monounsaturated VLCFA.     

Profiling and Imaging of Phospholipids in Brains of Abcd1‐Deficient Mice

ABCD1 is a gene responsible for X‐linked adrenoleukodystrophy (X‐ALD), and is critical for the transport of very long‐chain fatty acids (VLCFA) into peroxisomes and subsequent β‐oxidation. VLCFA‐containing lipids accumulate in X‐ALD patients, although the effect of ABCD1‐deficiency on each lipid species in the central nervous system has not been fully characterized. In this study, each phospholipid and lysophospholipid species in Abcd1‐deficient mice brains were profiled by liquid chromatography‐mass spectrometry. Among the phospholipid and lysophospholipid species that are significantly more enriched in Abcd1‐deficient mice brains, VLCFA were present in 75, 15, 5, 4, and 1 species of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylethanolamine, respectively. Most VLCFA were incorporated at the sn‐1 position of phosphatidylcholine and phosphatidylethanolamine. Among the phospholipid species that are significantly less enriched in Abcd1‐deficient mice brains, odd‐numbered saturated or mono‐unsaturated fatty acyl moieties are contained in all phosphatidylcholine species. In addition, a number of phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine species contained highly unsaturated fatty acyl moieties. Intriguingly, 44:1 phosphatidylcholine with VLCFA was mainly distributed in the gray matter, such as the cortex, but not in the white matter in the cerebrum and cerebellum. These results show that ABCD1‐deficiency causes metabolic alternation of long‐chain fatty acids and VLCFA. Moreover, our results imply a molecular mechanism for the incorporation of saturated or monounsaturated VLCFA into the sn‐1 position of phospholipids, and also indicate that the distribution of phospholipids with VLCFA may correlate with the development of X‐ALD.     

Lipid and fatty acid composition of diatoms revisited: rapid wound-activated change of food quality parameters influences herbivorous copepod reproductive success

Lipid and fatty acid composition are considered to be key parameters that determine the nutritive quality of phytoplankton diets for zooplanktonic herbivores. The fitness, reproduction and physiology of the grazers are influenced by these factors. The trophic transfer of lipids and fatty acids from algal cells has been typically studied by using simple extraction and quantification approaches, which, as we argue here, do not reflect the actual situation in the plankton. We show that cell disruption, as it occurs during a predator's grazing on diatoms can drastically change the lipid and fatty acid content of the food. In some algae, a rapid depletion of polyunsaturated fatty acids (PUFAs) is observed within the first minutes after cell disruption. This fatty acid depletion is directly linked to the production of PUFA-derived polyunsaturated aldehydes (PUA); these are molecules that are thought to be involved in the chemical defence of the algae. PUA-releasing diatoms are even capable of transforming lipids from other sources if these are available in the vicinity of the wounded cells. Fluorescent staining reveals that the enzymes involved in lipid transformation are active in the foregut of copepods, and therefore link the depletion processes directly to food uptake. Incubation experiments with the calanoid copepod Temora longicornis showed that PUFA depletion in PUA-producing diatoms is correlated to reduced hatching success, and can be compensated for by externally added single fatty acids.     

Varietal difference in lipid content and fatty acid composition of highbush blueberries

Lipids from five cultivars of highbush blueberries (Vaccinium corymbosum L.) were extracted and fractionated into neutral lipids (60–66%), glycolipids (20–22%) and phospholipids (14–18%). The major fatty acids in all fractions were palmitic (16∶0), oleic (18∶1), linoleic (18∶2), and linolenic (18∶3) acids. All lipid classes had a large concentration of C₁₈ polyunsaturated acids (84–92%), indicating that blueberries are a rich source of linoleic and linolenic acids. Changes in the fatty acid composition of neutral lipids and phospholipids were not significantly different among the five cultivars, but significant differences were noted in the ratios of linoleic and linolenic acids in the glycolipids fraction.     

Very long chain fatty acids in higher animals—A review

Fatty acids with greater than 22 carbon atoms (very long chain fatty acids, VLCFA) are present in small amounts in most animal tissues. Saturated and monoenoic VLCFA are major components of brain, while the polyenoic VLCFA occur in significant amounts in certain specialized animal tissues such at retina and spermatozoa. Biosynthesis of VLCFA occurs by carbon chain elongation of shorter chain fatty acid precursors while β-oxidation takes place, almost exclusively in peroxisomes. Mitochondria are unable to oxidize VLCFA because they lack a specific VLCFA coenzyme A synthetase, the first enzyme in the β-oxidation pathway. VLCFA accumulate in the tissues of patients with inherited abnormalities in peroxisomal assembly, and also in individuals with defects in enzymes catalyzing individual reactions along the β-oxidation pathway. It is believed that the accumulation of VLCFA in patient tissues contributes to the severe pathological changes which are a feature of these conditions. However, little is known of the role of VLCFA in normal cellular processes, and of the molecular basis for their contribution to the disease process. The present review provides an outline of the current knowledge of VLCFA including their biosynthesis, degradation, possible function and involvement in human disease.     

Effects of Cigarette Smoke on Cell Viability,Linoleic Acid Metabolism and Cholesterol Synthesis,in THP−1 Cells

Cigarette smoke (CS) contains thousands of substances, mainly free radicals that have as a target the polyunsaturated fatty acids (PUFA). Long chain PUFA are produced through elongation and desaturation reactions from their precursors; the desaturation reactions are catalyzed by different enzymes: the conversion of 18:2n-6 (linoleic acid, LA) to 18:3n-6 by Delta6 desaturase, while that of 20:3n-6 to 20:4n-6 by Delta5 desaturase. The aim of this work is to evaluate the effect of serum exposed to cigarette smoke (SE-FBS) on (1) cell viability and proliferation, (2) [1-(14)C] LA conversion and desaturase activities in THP-1 cells, a monocytic cell line. In THP-1, CS inhibits cell proliferation dose-dependently, by producing a modification in the cell cycle with a reduced number of cells in synthesis and mitosis phases at higher concentrations. CS also decreases [1-(14)C] LA conversion to its derivatives in a concentration-dependent manner, inhibiting the activities of Delta6 and mainly Delta5 desaturase. In addition, CS does not modify the incorporation of LA into various lipid classes but it reduces cholesterol synthesis from radiolabelled acetate, and increases free fatty acid, TG and CE levels. In conclusion, CS affects lipid metabolism, inhibiting LA conversion and desaturase activities. CS also shifts the "de novo" lipid synthesis from free cholesterol to TG and CE, where LA is preferentially esterified.     

Inhibition of Very Long Chain Fatty Acids Synthesis Mediates PI3P Homeostasis at Endosomal Compartments

A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant’s early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P₂ into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.     

Identification of n‐6 Monounsaturated Fatty Acids in Acer Seed Oils

Seed oils from Acer species are a potential source of the nutraceutical fatty acids, nervonic acid (cis‐15‐tetracosenoic acid, NA), and γ‐linolenic acid (cis‐6,9,12‐octadecatrienoic acid, GLA). To further characterize the genus, seed fatty acid content and composition were determined for 20 species of Acer. Fatty acid content ranged from 8.2% for Acer macrophyllum to over 36% for A. mono and A. negundo. The presence of very‐long‐chain fatty acids (VLCFA), with chain length of 20‐carbons or greater, and GLA were characteristic features of the seed oils. In all species, erucic acid (cis‐13‐docosenoic acid, EA) was the predominant VLCFA with the highest level of NA being only 8.6% in A. olivianum. Regioselective lipase digestion demonstrated that VLCFA are largely absent from the sn‐2 position of seed triacylglycerol, whereas GLA is primarily located at this position. Five Acer species contained low levels (<2%) of cis‐12‐octadecenoic acid and cis‐14‐eicosenoic acid, uncommon n‐6 fatty acids not previously reported from Acer.     

Biosynthesis of Long Chain Polyunsaturated Fatty Acids in the Marine Ichthyosporean Sphaeroforma arctica

Sphaeroforma arctica is a unique, recently discovered marine protist belonging to a group falling close to the yeast/animal border. S. arctica is found in cold environments, and accordingly has a fatty acid composition containing a high proportion of very long chain polyunsaturated fatty acids, including the ω3 polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA). Two elongases and five desaturases, representing the complete set of enzymes necessary for the synthesis of DHA from oleic acid, were isolated from this species and characterized in yeast. One elongase showed high conversion rates on a wide range of 18 and 20 carbon substrates, and was capable of sequential elongation reactions. The second elongase had a strong preference for the 20-carbon fatty acids EPA and arachidonic acid, with over 80 % of EPA converted to docosapentaenoic acid (DPA) in the heterologous yeast host. The isolation of a Δ8-desaturase, along with the detection of eicosadienoic acid in S. arctica cultures indicated that this species uses the alternate Δ8-pathway for the synthesis of long-chain polyunsaturated fatty acids. S. arctica also carried a Δ4-desaturase that proved to be very active in the production of DHA from DPA. Finally, a long chain acyl-CoA synthetase from S. arctica improved DHA uptake in the heterologous yeast host and led to an improvement in desaturation and elongation efficiencies.     

Production of C6–C14 Medium-Chain Fatty Acids in Seeds and Leaves via Overexpression of Single Hotdog-Fold Acyl-Lipid Thioesterases

ACYL-LIPID THIOESTERASES (ALT) are a type of plant acyl–acyl carrier protein thioesterase that generate a wide range of medium-chain fatty acids and methylketone (MK) precursors when expressed heterologously in Escherichia coli. While this makes ALT-type thioesterases attractive as metabolic engineering targets to increase production of high-value medium-chain fatty acids and MKs in plant systems, the behavior of ALT enzymes in planta was not well understood before this study. To profile the substrate specificities of ALT-type thioesterases in different plant tissue types, AtALT1-4 from Arabidopsis thaliana, which have widely varied chain length and oxidation state preferences in E. coli, were overexpressed in Arabidopsis seeds, Camelina sativa seeds, and Nicotiana benthamiana leaves. Seed-specific overexpression of ALT enzymes led to medium-chain fatty acid accumulation in Arabidopsis and Camelina seed triacylglycerols, and transient overexpression in N. benthamiana demonstrated that the substrate preferences of ALT-type thioesterases in planta generally agree with those previously determined in E. coli. AtALT1 and AtALT4 overexpression in leaves and seeds resulted in the accumulation of 12–14 carbon-length fatty acids and 6–8 carbon-length fatty acids, respectively. While it was difficult to completely profile the products of ALT-type thioesterases that generate MK precursors (i.e. β-keto fatty acids), our results nonetheless demonstrate that ALT enzymes are catalytically diverse in planta. The knowledge gained from this study is a significant step towards being able to use ALT-type thioesterases as metabolic engineering tools to modify the fatty acid profiles of oilseed crops, other plants, and microorganisms.     

How can we genetically engineer oilseed crops to produce high levels of medium‐chain fatty acids?

The end products of fatty acid synthase activities are usually 16‐ and 18‐carbon fatty acids. There are however, several plant species that store 8‐ to 14‐carbon (medium‐chain) fatty acids in their oil seeds. Among the medium‐chain fatty acids (MCFA), caprylic (8:0) and capric (10:0) are minor components of coconut oil, which are used in many industrial, nutritional and pharmaceutical products. Engineering crop plants such as Brassica could provide an economical source of these oils. During the last decade many laboratories have identified, cloned and characterized both the biosynthetic and catabolic enzymes regulating the composition and levels of these unusual fatty acids in seed oil. Among the biosynthetic enzymes thioesterases (TE), β‐ketoacyl‐ACP synthases (KAS) and acyltransferases are best characterized. In fact several independent investigators have shown that combined expression of the medium‐chain specific enzymes, specifically, TE, KAS and lysophosphatidic acid acyltransferase (LPAAT) results in the production of significant levels of MCFA in seed that otherwise do not accumulate any medium‐chain fatty acid. However, any additional increase in the levels of MCFA in transgenic seeds will require further detailed studies, such as possible induction of the medium‐chain specific enzymes in β‐oxidation and the glyoxylate pathways. To examine such a possibility, a number of genes involved in the β‐oxidation cycle among them a novel enzyme now designated as ACX3, a medium‐chain specific acyl‐CoA‐oxidase, has also been cloned. This article is an attempt to summarize our current knowledge and the present status of engineering oilseed crops for production of medium‐chain fatty acids.     

A delta9 desaturase from Bombus lucorum males: investigation of the biosynthetic pathway of marking pheromones

The knowledge of the molecular basis of communication in bumblebee communities is limited. None of the enzymes that participate in pheromone production have been characterized. Here, we cloned the gene encoding the Delta(9) desaturase from cDNA prepared from the total RNA of the pheromone gland and fat bodies of Bombus lucorum male. Functional expression of BlucNPVE desaturase in Saccharomyces cerevisiae and GC-MS analyses revealed its preference for C(18) saturated fatty acids. This suggests that Delta(9) desaturase is involved in the desaturation of metabolic fatty acids stored in triacylglyceroles (TAGs), because oleic acid is the most abundant fatty acid bound in TAG in B. lucorum and it is present in low concentration in the pheromone blend. The incubation of pheromone precursors with a dissected labial gland as well as direct injection of labelled pheromone substrates into B. lucorum males revealed that esterification of pheromone products occurs in the labial gland. These results support both the biosynthesis of pheromones from common lipids and the de novo synthesis of unsaturated pheromones in the labial gland.     

Identification and Characterization of Phospholipids with Very Long Chain Fatty Acids in Brewer’s Yeast

Yeast lipids and fatty acids (FA) were analyzed in Saccharomyces pastorianus from seven breweries and in the dietary yeast supplement Pangamin. GC–MS identified more than 30 FA, half of which were very‐long chain fatty acids (VLCFA) with hydrocarbon chain lengths of ≥22 C atoms. Positional isomers ω‐9 and ω‐7 were identified in FA with C18–C28 even‐numbered alkyl chains. The most abundant ω‐7 isomer was cis‐vaccenic acid. The structure of monounsaturated FA was proved by dimethyl disulfide adducts (position of double bonds and cis geometric configuration) and by GC–MS of pyridyl carbinol esters. Ultra‐high performance liquid chromatography‐tandem mass spectrometry with negative electrospray ionization identified the phospholipids phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine, with more than 150 molecular species. Wild‐type unmutated brewer's yeast strains conventionally used for the manufacture of food supplements were found to contain VLCFA.     

Molecular Species of Phospholipids with Very Long Chain Fatty Acids in Skin Fibroblasts of Zellweger Syndrome

The ratio of C_26:0/C_22:0 fatty acids in patient lipids is widely accepted as a critical clinical criterion of peroxisomal diseases, such as Zellweger syndrome and X-linked adrenoleukodystrophy (X-ALD). However, phospholipid molecular species with very long chain fatty acids (VLCFA) have not been precisely characterized. In the present study, the structures of such molecules in fibroblasts of Zellweger syndrome and X-ALD were examined using LC–ESI–MS/MS analysis. In fibroblasts from Zellweger patients, a large number of VLCFA-containing molecular species were detected in several phospholipid classes as well as neutral lipids, including triacylglycerol and cholesteryl esters. Among these lipids, phosphatidylcholine showed the most diversity in the structures of VLCFA-containing molecular species. Some VLCFA possessed longer carbon chains and/or larger number of double bonds than C_26:0-fatty acid (FA). Similar VLCFA were also found in other phospholipid classes, such as phosphatidylethanolamine and phosphatidylserine. In addition, VLCFA-containing phospholipid species showed some differences among fibroblasts from Zellweger patients. It appears that phospholipids with VLCFA, with or without double bonds, as well as C_26:0-FA might affect cellular functions, thus leading to the pathogenesis of peroxisomal diseases, such as Zellweger syndrome and X-ALD.     

Fatty Acyl Synthetases and Thioesterases in Plant Lipid Metabolism: Diverse Functions and Biotechnological Applications

Plants use fatty acids to synthesize acyl lipids for many different cellular, physiological, and defensive roles. These roles include the synthesis of essential membrane, storage, or surface lipids, as well as the production of various fatty acid-derived metabolites used for signaling or defense. Fatty acids are activated for metabolic processing via a thioester linkage to either coenzyme A or acyl carrier protein. Acyl synthetases metabolically activate fatty acids to their thioester forms, and acyl thioesterases deactivate fatty acyl thioesters to free fatty acids by hydrolysis. These two enzyme classes therefore play critical roles in lipid metabolism. This review highlights the surprisingly complex and varying roles of fatty acyl synthetases in plant lipid metabolism, including roles in the intracellular trafficking of fatty acids. This review also surveys the many specialized fatty acyl thioesterases characterized to date in plants, which produce a great diversity of fatty acid products in a tissue-specific manner. While some acyl thioesterases produce fatty acids that clearly play roles in plant-insect or plant-microbial interactions, most plant acyl thioesterases have yet to be fully characterized both in terms of their substrate specificities and their functions. The biotechnological applications of plant acyl thioesterases and synthetases are also discussed, as there is significant interest in these enzymes as catalysts for the sustainable production of fatty acids and their derivatives for industrial uses.     

Probing the compatibility of type II ketosynthase-carrier protein partners

Drug discovery often begins with the screening of large compound libraries to identify lead compounds. Recently, the enzymes that are involved in the biosynthesis of natural products have been investigated for their potential to generate new, diverse compound libraries. There have been several approaches toward this end, including altering the substrate specificities of the enzymes involved in natural product biosynthesis and engineering functional communication between enzymes from different biosynthetic pathways. While there exist assays to assess the substrate specificity of enzymes involved in these pathways, there is no simple method for determining whether enzymes from different synthases will function cooperatively to generate the desired product(s). Herein we report a method that provides insight into both substrate specificity and compatibility of protein-protein interactions between the acyl carrier protein (ACP) and ketosynthase (KS) domains involved in fatty acid and polyketide biosynthesis. Our technique uses a one-pot chemoenzymatic method to generate post-translationally modified ACPs that are capable of covalently interacting with KS domains from different biosynthetic systems. The extent of interaction between ACPs and KSs from different systems is easily detected and quantified by a gel-based method. Our results are consistent with previous studies of substrate specificity and ACP-KS binding interactions and provide new insight into unnatural substrate and protein interactions.     

Separation and quantitation of hydroxy and epoxy fatty acid by high-performance liquid chromatography with an evaporative light-scattering detector

A new high-performance liquid chromatography technique with an evaporative light-scattering detector (ELSD) has been developed for the separation and quantitative analysis of hydroxy and epoxy fatty acids. This method employs a gradual binary gradient (hexane/isopropanol) and ELSD detection. The minimum limit of detection is about 1 μg and ratio of mass to signal is essentially linear in the range of 10 to 200 μg. This high-performance liquid chromatography (HPLC) technique is able to separate various positional isomers of mono-hydroxy and dihydroxy fatty acids and can also discriminate between monohydroxy, epoxy, epoxyhydroxy, dihydroxy and trihydroxy fatty acids.     

α-氯代脂肪酸水热法中性水解合成α-羟基脂肪酸

开发了一条从天然脂肪酸合成α-羟基脂肪酸(α-HFA)的高产率、低污染且低成本的合成路线,即从天然脂肪酸制备α-氯代脂肪酸(α-CFA),再以α-CFA为原料通过水热法中性水解合成α-HFA。用GC-MS和IR鉴定了产物的结构,ESI-MS表征了产物的相对分子质量(以下简称分子量),纯化后α-HFA纯度达99%(GC)以上。探讨了水热中性水解反应中原料配比、反应加水量、反应温度、反应时间等因素对该水热工艺的影响,结果表明,当n(CaCO3)∶n(α-CFA)=1.05∶1,m(H2O)∶m(α-CFA)=2∶1,C12～C18的α-氯代天然脂肪酸在155℃水热条件下反应4 h后,α-HFA同系物的产率均高达98%～99%。     

Role of n-3 Fatty Acids on Bile Acid Metabolism and Transport in Dyslipidemia: A Review

Dietary n-3 fatty acids, especially of marine origin, eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3), have always been lauded for their profound effects on regulating the risk factors for major metabolic disorders. Yet, their consumption rate is poor compared to n-6 fatty acids [linoleic acid (18:2n-6)], which are predominantly consumed. Hence, the skewed n-6 to n-3 fatty acid ratio may have a bearing on the risk factors of various diseases, including dyslipidemia. Dyslipidemia and other lifestyle diseases associated with it, such as diabetes, obesity, hypertension, are a growing concern in both developed and developing countries. A common strategy for addressing dyslipidemia involves bile acid (BA) sequestration, to interrupt the enterohepatic circulation of BA, resulting in the modulation of lipid absorption in the intestine, thereby normalizing the levels of circulating lipids. The BA homeostasis is under the tight control of hepatic and enteric BA transporters. Many investigations have reported the effects of dietary constituents, including certain fatty acids on the reabsorption and transport of BA. However, a critical review of the effects of n-3 fatty acids on BA metabolism and transport is not available. The present review attempts to explore certain unmapped facets of the n-3 fatty acids on BA metabolism and transport in dyslipidemia, and their interplay with biological processes involving lipid rafts and gut microbiome.