Diacylglycerol Ethers Research Articles

Development and application of a pseudotargeted lipidomics method for alkylglycerol analysis

Alkylglycerols (1-O-alkyl-sn-glycerols) are microscale but critical lipids in foods. Conventional lipidomics analysis often loses sight of alkylglycerol analysis. In this study, we developed a high coverage pseudotargeted lipidomics method for analyzing alkylglycerols. The developed method integrated the advantages of GC–MS and LC–MS to profile alkylglycerol-type ether lipids comprehensively, with the help of a data processing Dart package termed FFIMA (Feature Fragments Information Matching Algorithm). The developed method exhibited competitive superiority to conventional lipidomics, such as wider coverage and higher accuracy. The validated method was assessed by three aquatic products and three milks. A total of 25 alkylglycerols, 107 diacylglycerol ethers, 21 monoacylglycerol ethers, 28 alkylglycerol-type ether phospholipids, and 35 plasmalogens were identified in the six foods. The results demonstrated that this method offers a comprehensive analysis of a wide spectrum of alkylglycerols.

Lipidomic profiling reveals biosynthetic relationships between phospholipids and diacylglycerol ethers in the deep-sea soft coral Paragorgia arborea

The cold-water gorgonian coral Paragorgia arborea is considered as a foundation species of deep-sea ecosystems in the northern Atlantic and Pacific oceans. To advance lipidomic studies of deep-sea corals, molecular species compositions of diacylglycerol ethers (DAGE), which are specific storage lipids of corals, and structural glycerophospholipids (GPL) including ethanolamine, choline, inositol and serine GPL (PE, PC, PI, and PS, respectively) were analyzed in P. arborea by HPLC and tandem mass spectrometry. In DAGE molecules, alkyl groups (16:0, 14:0, and 18:1), polyunsaturated fatty acids (PUFA), and monounsaturated FA are mainly substituted the glycerol moiety at position sn-1, sn-2, and sn-3, respectively. The ether form (1-O-alkyl-2-acyl) predominates in PE and PC, while PI is comprised of the 1,2-diacyl form. Both ether and diacyl forms were observed in PS. At position sn-2, C20 PUFA are mainly attached to PC, but C24 PUFA, soft coral chemotaxonomic markers, concentrate in PS, PI, and PE. A comparison of non-polar parts of molecules has shown that DAGE, ether PE, and ether PC can originate from one set of 1-O-alkyl-2-acyl-sn-glycerols. Ether PE may be converted to ether PS by the base-exchange reaction. A diacylglycerol unit generated from phosphatidic acid can be a precursor for diacyl PS, PC, and PI. Thus, a lipidomic approach has confirmed the difference in biosynthetic origins between ether and diacyl lipids of deep-sea gorgonians.

Enrichment of Alkylglycerols and Docosahexaenoic Acid via Enzymatic Ethanolysis of Shark Liver Oil and Short-path Distillation

ABSTRACT Enzymatic alcoholysis of squalene-free shark liver oil (SLO) for the enrichment of alkylglycerols (AKG) and docosahexaenoic acid (DHA) was investigated. The squalene-free SLO contained 75.11% triacylglycerols (TAG) and 18.69% diacylglycerol ethers (DAGE), and its DHA and total AKG contents were 7.52% and 6.17%, respectively. The catalytic discrimination against both DAGE and DHA was observed during the reaction, and the discrimination against DAGE was affected by lipase and reaction conditions. The ethanolysis reaction was carried out at 40°C for 24 h, where the reaction medium contained 20% ethanol and 3% Lipozyme® 435. The ethanolysis derivatives with 62.96% monoacylglycerol ethers, 30.93% monoacylglycerols, and 5.88% DAGE were obtained followed by short-path distillation, and its DHA and total AKG contents were improved to 23.17% and 33.98%, respectively. The study provides an approach for value-added utilization of SLO with low contents of AKG and n-3 polyunsaturated fatty acids.

Triacylglycerol estolides, a new class of mammalian lipids, in the paracloacal gland of the brushtail possum (Trichosurus vulpecula).

The paracloacal glands are the most prevalent scent glands in marsupials, and previous investigation of their secretions in the brushtail possum (Trichosurus vulpecula) has identified many odorous compounds together with large amounts of neutral lipids. We have examined the lipids by LC-MS, generating ammonium adducts of acylglycerols by electrospray ionisation. Chromatograms showed a complex mixture of coeluting acylglycerols, with m/z from about 404 to 1048. Plots of single [M + NH4](+) ions showed three groups of lipids clearly separated by retention time. MS-MS enabled triacylglycerols and diacylglycerol ethers to be identified from neutral losses and formation of diacylglycerols and other product ions. The earliest-eluting lipids were found to be triacylglycerol estolides, in which a fourth fatty acid forms an ester link with a hydroxy fatty acid attached to the glycerol chain. This is the first report of triacylglycerol estolides in animals. They form a complex mixture with the triacylglycerols and diacylglycerol ethers of lipids with short- and long-chain fatty acids with varying degrees of unsaturation. This complexity suggests a functional role, possibly in social communication.

Discrimination against diacylglycerol ethers in lipase-catalysed ethanolysis of shark liver oil

Lipase-catalysed ethanolysis of squalene-free shark liver oil was investigated. The mentioned shark liver oil was comprised mainly of diacylglycerol ether and triacylglycerols. In order to test discrimination against diacylglycerol ether, up to 10 different lipases were compared. The ratio of oil to ethanol and lipase stability were also evaluated. Surprisingly, lipase from Pseudomonas stutzeri was the fastest biocatalyst among all assayed, although poor discrimination against diacylglycerol ether was observed. The best results in terms of selectivity and stability were obtained with immobilised lipase from Candida antarctica (Novozym 435). Ethanolysis reaction after 24h in the presence of Novozym 435 produced total disappearance of triacylglycerol and a final reaction mixture comprised mainly of diacylglycerol ethers (10.6%), monoacylglycerol ethers (32.9%) and fatty acid ethyl esters (46.0%). In addition, when an excess of ethanol was used, diacylglycerol ethers completely disappeared after 15h, giving a final product mainly composed of monoacylglycerol ethers (36.6%) and fatty acid ethyl esters (46.4%).

Chemoenzymatic synthesis of a focused library of enantiopure structured 1- O-alkyl-2,3-diacyl- sn-glycerol type ether lipids

A highly efficient two-step chemoenzymatic synthesis of enantiopure structured ether lipids of the 1- O-alkyl-2,3-diacyl- sn-glycerol type has been developed. Chimyl, batyl and selachyl alcohols possessing pure saturated fatty acid (SFA) attached to the sn-3 position and pure EPA and DHA attached to the sn-2 position were obtained under full regiocontrol. This was offered by mild conditions and a highly efficient lipase that operated at room temperature. High-resolution 1H NMR spectroscopy was used to monitor the progress of the reactions and to evaluate the full regiocontrol of the reactions involved by keeping track of all prospective adducts involved in these reactions. This was extended to preparation of a focused library of eight monoacyl intermediate adducts for all even-numbered SFA ranging from C 2–C 16 and the corresponding EPA and DHA structured diacyl glycerol ethers (DAGE) products for chimyl, batyl and selachyl alcohols, the total of 72 compounds.

Improved separation and quantification of neutral and polar lipid classes by HPLC–ELSD using a monolithic silica phase: Application to exceptional marine lipids

An improved HPLC method is presented, which allows separation and quantification of a broad range of lipid classes of marine zooplankton with special regard to neutral lipids. Marine zooplankton species often produce high amounts of exceptional lipids, especially at high latitudes, in order to cope with the harsh environmental conditions and strong seasonality in food supply. Major neutral lipid classes are wax esters, triacylglycerols, diacylglycerol ethers, free fatty alcohols and sterols. Neutral and polar lipids were separated and identified on a monolithic silica column (Chromolith ®Performance-Si) using high performance liquid chromatography (HPLC) with an evaporative light scattering detector (ELSD). The method resolves a broad spectrum of lipids, varying in polarity from squalene to lysophosphatidylcholine in a single run. The total run time was 35 min including column re-equilibration. The calibration was made at levels of 0.1–60 μg lipid/injection, but a 10–15-fold greater amount can be injected if single lipid classes need to be separated, e.g. for further determination of individual fatty acids. The method was applied to representative Arctic zooplankton species (copepods, pteropods, euphausiids and ctenophores) that are known to biosynthesize in particular neutral lipids like diacylglycerol ethers and free fatty alcohols.

Trophic relationships of white-chinned petrels from Crozet Islands: combined stomach oil and conventional dietary analyses

The diet of white-chinned petrels Procellaria aequinoctialis breeding at the Crozet Archipelago (southern Indian Ocean) was studied using two complementary methods: lipid analysis of stomach oils as trophic markers together with the conventional dietary approach (i.e., stomach content analysis). Objectives were (1) to investigate the adult diet when they feed for themselves by analyzing stomach oil lipids, and (2) to compare the lipid signature of chick and adult oils. Stomach oils mainly consisted of triacylglycerols (TAG), diacylglycerol-ethers (DAGE) and wax esters (WE) (66, 14 and 11%, respectively). The dietary origin of TAG and WE was evaluated by linear discriminant analyses with fatty acid and fatty alcohol fractions. Analyses evidenced that stomach oils did not originate from Antarctic krill, but instead from myctophid fish, thus demonstrating the importance of mesopelagic fish in the nutrition of adult petrels. This result was consistent with the identification of digested remains of myctophids recovered from adult stomach contents after long foraging trips. Large amounts of a rare lipid class, DAGE (up to 76% of total lipids), were identified in two stomach oils, together with fresh remains of the squid Gonatus antarcticus (99% by mass), suggesting that DAGE could have the potential to be trophic markers of cephalopods. Moreover, six oils probably originated from Patagonian toothfish, thus confirming strong interactions between white-chinned petrels and fisheries. Comparison between chick and adult stomach oils indicated no major differences in their biochemical composition suggesting an identical dietary origin of oils, mainly myctophids. Both adult and chick oils can therefore be used to determine the feeding ecology of adult birds when they feed far away from their breeding grounds. Finally, food analysis of chick samples and adult samples collected after short and long trips indicated different foraging grounds during the two kinds of trips, and also between long trips performed in subtropical and Antarctic waters.

Exceptional long-term starvation ability and sites of lipid storage of the Arctic pteropod Clione limacina

The Arctic pteropod Clione limacina was collected in Kongsfjorden, Svalbard, in mid June 2004, to study the lipid metabolism within the sites of lipid storage structures during long-term starvation. Animals survived in an aquarium without any food for nearly 1 year (356 days). Size, number of lipid droplets, dry and lipid mass, lipid class and fatty acid compositions of C. limacina were determined and separately analysed for the digestive gland and the remaining integument. During the starvation period, animals shrunk from 22.4 to 12 mm in length on average, and the number of lipid droplets decreased from 1,600 to 1,000 per animal. Dry mass (DM) and total lipid mass both dropped by about 80% from day 200 to the end. The lipid content as percentage DM of the total organism did not decrease significantly ranging from 43.8 to 32.3%DM. The lipid content of the trunk was moderate with about 20%DM. The digestive gland was very rich in lipids with more than 70%DM throughout the experiment and is the major site of lipid metabolism and storage. Triacylglycerols (TAG) decreased, in the total organism, from high initial levels of 62.6 to 43% of total lipid at the end. In contrast, the proportions of 1-O-alkyldiacylglycerols [diacylglycerol ethers (DAGE)] remained almost constant, varying between 20.4 and 28.4%. In the digestive gland, TAG ranged from 60.3 to 64.8% and DAGE from 23.6 to 32.2% from day 200 to the end of the experiment. TAG and DAGE of the trunk were most likely located in the lipid droplets and were almost depleted at the end of starvation. Besides their function as lipid deposit DAGE may also act as protecting substance against bacterial and fungal infections. During the first 200 days of starvation, the fatty acid compositions showed only small variations. Thereafter, fatty acids typical for storage lipids decreased in all body compartments. In adaptation to long periods of food scarcity, C. limacina has evolved various strategies as body shrinkage, utilisation of body constituents not essential for survival, a very low metabolism and slow lipid consumption.

Lipid storage in marine zooplankton

Zooplankton storage lipids play an important role during reproduction, food scarcity, ontogeny and diapause, as shown by studies in various oceanic regions. While triacylglycerols, the primary storage lipid of terrestrial animals, are found in almost all zooplankton species, wax esters are the dominant storage lipid in many deep-living and polar zooplankton taxa. Phospholipids and diacylglycerol ethers are the unique storage lipids used by polar euphausiids and pteropods, respec- tively. In zooplankton with large stores of wax esters, triacylglycerols are more rapidly turned over and used for short-term energy needs, while wax esters serve as long-term energy deposits. Zooplankton groups found in polar, westerlies, upwelling and coastal biomes are characterized by accumulation of large lipid stores. In contrast, zooplankton from the trades/tropical biomes is mainly composed of omnivorous species with only small lipid reserves. Diapausing copepods, which enter deep water after feeding on phytoplankton during spring/summer blooms or at the end of upwelling periods, are characterized by large oil sacs filled with wax esters. The thermal expansion and com- pressibility of wax esters may allow diapausing copepods and other deep-water zooplankton to be neutrally buoyant in cold deep waters, and they can thus avoid spending energy to remain at these depths. Lipid droplets are often noted in zooplankton ovaries, and a portion of these droplets can be transferred to developing oocytes. In addition to lipid droplets, zooplankton eggs have yolks with lipovitellin, a lipoprotein with approximately equal amounts of protein and lipid. The lipovitellin lipid is predominantly phosphatidylcholine, so during reproduction females must convert a portion of their storage lipid into this phospholipid. Developing embryos use their lipovitellin and lipid droplets for energy and materials until feeding begins. The various functions storage lipids serve during the dif- ferent life history stages of zooplankton are very complex and still not fully understood and hence offer a multitude of fascinating research perspectives.

Exceptional lipids and fatty acids in the pteropod Clione limacina (Gastropoda) from both polar oceans

Abstract The gymnosomatous and thecosomatous pteropods, Clione limacina and its only prey Limacina helicina and L. retroversa , from Arctic and Antarctic waters as well as particulate matter have been investigated with respect to their lipid compositions. The close trophic relationships between phytoplankton– Limacina – Clione were partially reflected by the fatty acid compositions due to high amounts of 16:1( n −7), which indicate the ingestion of diatoms in L. helicina . However, the outstanding feature of this food chain concerns the extraordinary lipid and fatty acid compositions of C. limacina . Up to 40.6% of its total lipid consisted of 1- O -alkyldiacylglycerol ethers, a very unusual depot lipid in the zooplankton. The other major storage lipid were triacylglycerols, which occurred in similar amounts. In addition, the various lipid classes of C. limacina exhibited unique fatty acid compositions, due to high percentages of odd-chain length components, up to one third of total fatty acids. The dominant odd-chain fatty acid was 17:1( n −8) reaching 18%, followed by 15:0 and 17:0, with 17:1( n −8) enriched in the diacylglycerol ethers. Major even-chain fatty acids were 16:0, 16:1( n −7), 20:5( n −3) and 22:6( n −3). In L. helicina neither diacylglycerol ethers nor odd-chain fatty acids could be detected, indicating that these atypical lipids are not ingested with the prey by C. limacina . De novo biosynthesis of these compounds is therefore suggested. The synthesis of odd-chain fatty acids is initiated with propionate. This precursor may originate from dimethyl- β -propiothetin (DMPT), which has been described to be accumulated by L. helicina via phytoplankton uptake. Hence, C. limacina not only represents an exceptional member of the polar zooplankton due to its extreme trophic specialisation, it has also developed exceptional lipid biochemical adaptations in the Arctic and Antarctic ocean.

Capillary supercritical fluid chromatographic analysis of shark liver oils

AbstractThe liver oils of six different shark species have been analyzed by supercritical fluid chromatography (SFC). The liver oils were from the species Pseudotriakis microdon (False catshark), Centroscymnus coelepsis (Portuguese dogfish), Centrophorus squanosus (Leafscalp gulper shark), Deanea calceus (Birdbeak dogfish), Etmopterus princips (Greater lantern shark), and Centroscymnus crepidater (Longnose velvet dogfish). The method was capable of direct quantitation of squalene and cholesterol, while quantitation of triacylglycerols, cholesterol esters, and diacylglycerol ethers required thin‐layer chromatographic fractionation prior to SFC analysis. The iodine values of the liver oil samples gave a linear correlation when plotted against the squalene content found by SFC. The variation of squalene content within one shark species is large, and there are large differences in squalene content from species to species. The squalene contents varied between 0.22 and 82.54 wt%. The identity of the glycerol ethers was investigated by SFC of the unsaponifiable matter. The major glycerol ethers contained chimyl, batyl, and selachyl alcohol.

Digestion of the 1-O-alkyl diacylglycerol ethers of Atlantic dogfish liver oils by Atlantic salmon Salmo salar.

Dogfish (Squalus acanthias) liver poses a waste disposal problem in Canada because it is not utilized for any commercial purpose. The liver of Atlantic dogfish, which is often up to 20% of the weight of the fish, contains 40-70% oil. The oil contains about 30-40% 1-O-alkyl diacylglycerol ethers (DAGE) which render it unacceptable for human use, and it has also not been considered satisfactory for animal feed use. Polyunsaturated fatty acids (20:5n-3 and 22:6n-3) are present in dogfish liver oils at levels comparable to those in herring oil. Dogfish liver oil could be a source of essential fatty acids for Atlantic salmon (Salmo salar), but their ability to hydrolyze DAGE from dogfish oil has not been examined. Experiments were designed to measure the digestibility of fatty acids of DAGE in salmon. The fatty acid moieties were liberated by the digestive enzymes of the fish and made readily available as a source of energy. The 1-O-alkylglycerol ether moiety was absorbed to a small extent but should not constitute a health problem in either the fish or the human fish consumer. The long-chain polyunsaturated fatty acids were particularly well absorbed, with an apparent digestibility in salmon of 87-95% when feeding on dogfish liver oil. The total fatty acids and other lipids were in fact both absorbed to the extent of approximately 85%.

Lipid and fatty acid characterization and metabolism in the sea anemonePhymactis clematis (Dana)

AbstractNeutral lipid, phospholipids and fatty acids of the sea anemonePhymactis clematis from the south‐west Atlantic were characterized and quantified in spring and autumn. Neutral lipids predominated over phospholipids in both seasons. Triacylglycerol and diacylglycerol ethers were the major lipids. In spring, an increase of esterified sterols was noted. The major fatty acids found were 22∶5ω3, 20∶5ω3 and 16∶0. The sea anemones were also incubated in vivo with either [1‐14C]linoleate or [1‐14C] α‐linolenate for 2 hr. Isotope incorporation into lipids and their transformations into higher fatty acids were examined. Both precursors were incorporated into the lipids, mainly in triacylglycerols and mono‐acylglycerols, while α‐linolenate was also incorporated into phospholipids. The radioactive linoleate was elongated to 20∶2, 22∶2 and 24∶2 fatty acids, but not desaturated to 18∶3ω6. α‐Linolenate was desaturated by Δ6 desaturase to 18∶4ω3. The specificity of Δ6‐desaturase is discussed.

Effect of the environment and fasting on the lipid and fatty acid composition ofDiplodom patagonicus.

Some effects of food, habitat and temperature on the lipid composition of a freshwater mollusk,Diplodom patagonicus, were studied. Animals kept and fasted up to 60 days in an aquarium at 9 C and 20 C showed a decrease of the total lipid content that corresponded to a decrease of triacylglycerols and diacylglycerol ethers. This decrease evoked an increase of polar-to-nonpolar lipid ratio. However, no significant change in the total fatty acid composition was shown. Moreover, a decrease of temperature from 20 C to 9 C decreased the incorporation of labeled linoleic and α-linolenic acid into the lipids, but did not modify the unsaturated: saturated acid ratio of the mollusk lipids during this period. A change of habitat from lake to estuary changed very significantly the fatty acid composition of the animal. The ω6 acids, linoleic and arachidonic, typical ofD. patagonicus living in the lake, were partially replaced by ω3 acids. That this change was due to a change of food composition was indicated by the fatty acids of corresponding sediments. Therefore, the fatty acid composition ofD. patagonicus is highly sensitive to food composition and varies little with temperature and seasonal changes.

The identification of ceramides and glyceryl ethers in unsaponifiable lipid of human aorta

Unsaponifiable material from a lipid fraction isolated from atherosclerotic human aorta, contained cerebrosides, which had been previously identified, ceramides, and glycerol ethers, as well as other, still unidentified, substances. Identification of the ceramides and glycerol ethers was made primarily by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. Ceramides were present with the fatty acids 16:0, 23:0, 24:0, and 24:1. Other fatty acids were found which were probably primarily associated with ceramides. These acids were 18:0, 18:1, 20:0, 22:0, and 25:0. The glycerol ethers were 1-glycerol ethers of the fatty alcohols 16:0, 18:0, and 18:1. Glycerol ethers may well have been formed from diacylglycerol ethers during the isolation procedure.

The densities of three classes of marine lipids in relation to their possible role as hydrostatic agents.

AbstractWax esters, diacyl glycerol ethers and triglycerides were isolated from marine oils by preparative thin layer chromatography. Their densities indicated that wax esters would nearly equal squalene in effectiveness as a bouyant agent and that diacyl glycerol ethers would surpass triglycerides. Their compositions were listed to permit comparison with lipids from other sources.

Studies on the stomach oils of marine animals—I. Oils of the black shark Dalatias licha (Bonnaterre)

1. 1. Eight specimens of Dalatias licha contained between 12 ml and 550 ml of oil in their stomachs. 2. 2. Squalene, wax esters, diacyl glycerol ethers and triglycerides were major components of the stomach oils. 3. 3. The stomach oils are believed to be dietary residues resulting from inadequate gastric peristalsis. 4. 4. The large quantities of squalene in the liver oils appear to be of dietary origin.

Metabolism of glycerol ether-containing lipids in dogfish (Squalus acanthias)

Dogfish (Squalus acanthias) received intrahepatic injections of either palmitic acid-1-14C or chimyl alcohol-1-14C. The lipids of the liver were then analyzed for incorporated radioactivity. The experiments with labeled palmitic acid demonstrated that fatty acids are reductively incorporated into the alkyl and alkenyl ether chains of glycerolipids. Significantly lower specific activities were found for the diacyl alk-1′-enyl ethers and diacyl glycerol ethers than for other glycerol ether-containing lipids. These compounds may therefore represent terminal points in ether-lipid metabolism. The studies with labeled chimyl alcohol indicate that dogfish liver contains enzymes that have a high capacity for oxidatively cleaving alkyl ether linkages. Furthermore, it is probable that alkyl ethers are converted directly to alkenyl ethers, possibly via a biodehydrogenation reaction.