Lipidomics of the sea sponge Amphimedon queenslandica and implication for biomarker geochemistry

Demosponges are a rich natural source of unusual lipids, some of which are of interest as geochemical biomarkers. Although demosponges are animals, they often host dense communities of microbial symbionts, and it is therefore unclear which lipids can be synthesized by the animal de novo, and which require input from the microbial community. To address this uncertainty, we analyzed the lipids of Amphimdeon queenslandica, the only demosponge with a published genome. We correlated the genetic and lipid repertoires of A. queenslandica to identify which biomarkers could potentially be synthesized and/or modified by the sponge. The fatty acid profile of A. queenslandica is dominated by an unusual Δ^5,9 fatty acid (cis‐5,9‐hexacosadienoic acid)—similar to what has been found in other members of the Amphimdeon genus—while the sterol profile is dominated by C₂₇‐C₂₉ derivatives of cholesterol. Based on our analysis of the A. queenslandica genome, we predict that this sponge can synthesize sterols de novo, but it lacks critical genes necessary to synthesize basic saturated and unsaturated fatty acids. However, it does appear to have the genes necessary to modify simpler products into a more complex “algal‐like” assemblage of unsaturated fatty acids. Ultimately, our results provide additional support for the poriferan affinity of 24‐isopropylcholestanes in Neoproterozoic‐age rocks (the “sponge biomarker” hypothesis) and suggest that some algal proxies in the geochemical record could also have animal contributions.     

Effect of exogenous sterols on the growth and fatty acid composition of the oomycete Pythium debaryanum

Exogenous ergosterol and cholesterol were found to affect the growth and lipogenesis of the oomycete fungus Pythium debaryanum, which is unable to synthesize de novo steroid compounds. These sterols stimulated the growth of the fungus during its submerged cultivation in glucose-peptone medium. This was accompanied by the shortening of the lag phase, the lengthening of the period of active growth, and by a 3.7- or 4.3-fold increase in the maximum biomass in response to the addition of ergosterol or cholesterol, respectively. In the presence of ergosterol, the cellular content of polyenoic fatty acids increased, and the relative content of eicosapolyenoic fatty acids reached 31.4% of the total amount of fatty acids in cells. Conversely, cholesterol decreased the cellular content of polyenoic acids, and the relative content of eicosapolyenoic acids fell to 19.6% of the total amount of fatty acids. It may be inferred that exogenous sterols enhance the yield of pharmacologically active polyenoic acids because of the growth stimulation.     

Effect of exogenous sterols on the growth and fatty acid composition of the oomycetePythium debaryanum

Exogenous ergosterol and cholesterol were found to affect the growth and lipogenesis of the oomycete fungusPythium debaryanum, which is unable to synthesize de novo steroid compounds. These sterols stimulated the growth of the fungus during its submerged cultivation in glucose-peptone medium. This was accompanied by the shortening of the lag phase, the lengthening of the period of active growth, and by a 3.7-or 4.3-fold increase in the maximum biomass in response to the addition of ergosterol or cholesterol, respectively. In the presence of ergosterol, the cellular content of polyenoic fatty acids increased, and the relative content of eicosapolyenoic fatty acids reached 31.4% of the total amount of fatty acids in cells. Conversely, cholesterol decreased the cellular content of polyenoic acids, and the relative content of eicosapolyenoic acids fell to 19.6% of the total amount of fatty acids. It may be inferred that exogenous sterols enhance the yield of pharmacologically active polyenoic acids because of the growth stimulation.     

Fatty acid composition from an epiphytic strain of Fusarium oxysporum associated with algal crusts

The fatty acid composition from Fusarium oxysporum B1, a fungal epiphyte from algal crusts, was determined. The most prominent fatty acids found in the mycelium were 18:1 omega9c, 16:0 and 18:0. These compounds amounted 47% of total fatty acids, whereas remaining fatty acids identified, including beta-hydroxylated, iso-branched, diunsaturated and odd-numbered derivatives were present in minor amounts.     

An investigation of the mechanisms for sterol synthesis and dietary sterol bioconversion in the heterotrophic protists Oxyrrhis marina and Gyrodinium dominans

The ability of the marine heterotrophic protists Oxyrrhis marina and Gyrodinium dominans to synthesize sterols de novo and modify dietary sterols was investigated using ¹³C-labeled substrates. De novo sterol synthesis of O. marina was determined by incorporation of ¹³C acetate into the culture medium. For G. dominans which has low tolerance of acetate, a protozoan prey Perkinsus marinus that cannot synthesize sterols, was cultured with ¹³C acetate then fed to G. dominans. Both heterotrophs utilized dietary ¹³C to synthesize fatty acids de novo, but not sterols. The ability of O. marina and G. dominans to alkylate, saturate, and desaturate dietary sterols was tested using P. marinus incorporated with ¹³C-labeled cholesterol as prey. O. marina did not modify the dietary ¹³C-cholesterol, but G. dominans produced 5 labeled sterols (brassicasterol, C28:1, and unknown C28, C29 and C30 sterols) indicating that G. dominans has the ability to desaturate and alkylate dietary cholesterol. The ability of O. marina and G. dominans to dealkylate dietary sterols was tested by feeding them gelatin acacia microspheres (GAMs) containing ¹³C-labeled brassicasterol. Neither heterotroph dealkylated brassicasterol to make cholesterol, but G. dominans alkylated and saturated brassicasterol to make 2 sterols (C29:1 and C30:0). The lack of dealkylation of brassicasterol by both protist species suggests problems with the substrate and/or delivery system since previous studies suggest that dealkylation of brassicasterol occurs when either species is fed algae containing this sterol.     

Reassessment of the role of phospholipids in sexual reproduction by sterol-auxotrophic fungi.

Several genera of oomycete fungi which are incapable of de novo sterol synthesis do not require these compounds for vegetative growth. The requirement for an exogenous source of sterols for sexual reproduction by several members of the Pythiaceae has been questioned by reports of apparent induction and maturation of oospores on defined media supplemented with phospholipids in the absence of sterols. A more detailed examination of this phenomenon suggested that trace levels of sterols in the inoculum of some pythiaceous fungi act synergistically with phospholipid medium supplements containing unsaturated fatty acid moieties to induce oosporogenesis. Phospholipid analysis of one species, Pythium ultimum, suggested that only the fatty acid portion of the exogenous phospholipid is taken up by the fungus. Enrichment of the phospholipid fraction of total cell lipid of P. ultimum with unsaturated fatty acids promoted oospore induction, and enhanced levels of unsaturated fatty acids in the neutral lipid fraction increased oospore viability. For some pythiaceous fungi, the levels of sterols required for the maturation of oospores with appropriate phospholipid medium supplementation suggest that these compounds are necessary only for the sparking and critical domain roles previously described in other fungi.     

Isoprenoid biosynthesis in a marine sponge of the Amphimedon genus: incorporation studies with [1-14C] acetate, [4-14C] cholesterol and [2-14C] mevalonate

1. We present quantitative evidence from incorporation of [1-14C] acetate that the enzymes to synthesise isoprenoids are present in the marine sponge Amphimedon sp. and that efficient carotenoid synthesis takes place. 2. The de novo synthesis of b,b-carotene and (3R,3'R)-zeaxanthin may occur in a chlorophyll a-producing microalgal symbiont with subsequent aromatisation to (3R)-isoagelaxanthin by the sponge itself. 3. Amphimedon sp. contains nuclear-modified sterols derived by modification of conventional dietary sterols.     

Lipid Composition of the Marine Ciliates Pleuronema sp. and Fabrea salina: Shifts in Response to Changes in Diet1

ABSTRACT. The composition and incorporation of lipids in two marine ciliates, Pleuronema sp. and Fabrea salina, was examined following growth on either an algal or bacterial diet. When allowed to feed on a natural bacterial community, Pleuronema sp. synthesized the triterpenoid alcohol gammaceran-3β-ol (tetrahymanol) and two hopanoids (hopan-3β-ol and one uncharacterized hopanoid). When fed the marine alga Isochrysis galbana, F. salina contained the major algal sterol 24-methylcholesta-5, 22-dien-3β-ol and several long chain ketones specific to the alga. In both ciliates, fatty acids composition showed a general correspondence to that of the diet. Using a series of antibiotic treatments to alter the bacterial prey community, and thus fatty acid composition of the ciliate's diet, promoted changes in the fatty acid composition of Pleuronema sp. to resemble that of the bacterial prey. The addition of a mixture of algal sterols to a bacterized culture of another scuticociliate, Parauronema acutum, inhibited tetrahymanol synthesis and resulted in the incorporation of sterols into the ciliate.     

Lipidomics of hepatic lipogenesis inhibition by omega 3 fatty acids

We assessed – by a lipidomic approach – the differential incorporation of EPA and DHA into hepatic lipids, after prolonged feeding of rats with fish oil. We also evaluated their effect on lipogenesis and its related enzymes. Rats were administered 100 mg/kg/d fish oil, by oral gavage, for 30 days. The fatty acid profile of total liver lipids was determined by gas–liquid chromatography coupled to mass spectrometry. Individual phospholipid classes and their molecular species were quantified by ESI-MS/MS. Omega 3 fatty acids readily incorporated into hepatic phospholipids, decreased stearoyl-CoA desaturase 16, stearoyl-CoA desaturase, delta 6 desaturase, and delta 5 desaturase activities (calculated as product/substrate ratio) and decreased the “lipogenesis index”, i.e., the proportion of fatty acids endogenously synthesized in the liver and not provided with the diet. Our results show that long-chain omega 3 fatty acids selectively incorporate into hepatic phospholipids, inhibit de novo lipogenesis and change the hepatic fatty acid profile via reduced desaturases' activity in the non-steatotic liver. In addition to corroborating advice to consume adequate amounts of omega 3 fatty acids for overall health, these data contribute mechanistic insights to the clinical observations that provision of omega 3 fatty acids decreases hepatic fat and ameliorates NAFLD prognosis.     

Lipids of the Spirochaetales: Comparison of the Lipids of Several Members of the Genera Spirochaeta, Treponema, and Leptospira

The lipid compositions of 17 spirochetes belonging to the genera Spirochaeta and Treponema were investigated and compared with data previously derived from 11 strains of Leptospira. The lipid compositions and lipid metabolism of any of these genera is sufficiently different to be characteristic of that genus and to differentiate it from the other two genera. Members of the genus Leptospira are characterized by their ability to beta-oxidize long chain fatty acids as their major carbon and energy source. With few exceptions, they are incapable of synthesizing fatty acids de novo. The major phospholipid found was phosphatidyl ethanolamine. No glycolipid or phosphatidyl choline was found in these organisms. Members of the genus Treponema studied were incapable of beta-oxidation as well as de novo synthesis of fatty acids. Phosphatidyl choline is the major phospholipid of this genus. The glycolipid, monogalactosyl diglyceride, is a major component of the Treponema. Members of the Spirochaeta did synthesize fatty acids de novo. Although these spirochetes contain a monoglycosyl diglyceride, the hexose content of the glycolipid varied from species to species. Neither phosphatidyl ethanolamine nor phosphatidyl choline was found in the Spirochaeta.     

Lipids of the Green Alga BotryococcusCultured in a Batch Mode

The lipid fraction of the green alga Botryococcuscultured in a batch mode was found to contain polar lipids (more than 50% of the total lipids), di- and triacylglycerols, sterols and their esters, free fatty acids, and hydrocarbons. In aging culture, the content of polar lipids somewhat decreased and that of triacylglycerols increased by more than four times. The content of hydrocarbons in the algal biomass did not exceed 0.9% and depended little on the culture age. Intracellular lipids contained saturated and unsaturated (mono-, di-, and trienoic) fatty acids. The maximum content of C_{16 : 3}and -C_{18 : 3}fatty acids (up to 35% of the total fatty acids) was detected in the phase of active growth. The extracellular and intracellular lipids of the alga differed in the proportion of particular lipids and in the fatty acid pattern.     

Changes in apolar metabolites during in vitro organogenesis of Pancratium maritimum

Calli, shoot-clumps and regenerated plants were initiated from young fruits of Pancratium maritimum L. Their genetic stability was monitored by flow cytometry before chemical studies. Apolar metabolites (alkaloids extracted at pH > 7, free fatty acids and fatty alcohols, sterols etc.) were qualitatively and quantitatively analyzed by GC–MS. The results clearly demonstrated that alkaloid synthesis in P. maritimum is closely related with tissue differentiation. The highest amounts of alkaloids and presence of homolycorine and tazettine type compounds (end products of the biosynthetic pathway of the Amaryllidaceae alkaloids) were found in highly differentiated tissues. Galanthamine accumulated in the leaves of plantlets. The amount of hordenine, a protoalkaloid, is related with the ability of tissues to synthesize alkaloids. Saturated fatty acids were found in considerably higher levels in undifferentiated callus cultures and partially differentiated shoot-clumps than in regenerated plants. Mono- and dienoic fatty acids were found at higher levels in non-photosynthesizing tissues – calli, and in vitro and intact bulbs, while α-linolenic acid (trienoic acid) was found in higher amounts in the photosynthesizing leaves of shoot-clumps and regenerated plants than in bulbs and calli. Fatty alcohols were found mainly in leaves, while sterols tended to accumulate in photosynthesizing and undifferentiated tissues.     

Rat liver nuclear lipids. Composition and biosynthesis

A characteristic of rat liver nuclear lipids is their high content in neutral lipids especially of tryglycerides and free fatty acids. These compounds do not arise due to hydrolysis of more complex lipids during the isolation of the nuclei. The neutral lipids fatty acid pattern is more saturated than the phospholipid one. The presence of phosphatidylinositol mono- and diphosphate in nuclei has been confirmed. Nuclei are unable to synthesize de novo phospholipids but are capable of incorporating inorganic phosphate into lipids synthesized via a kinase pathway.     

Biosynthesis of fatty acids and triacylglycerols by 2,6,10,14-tetramethyl pentadecane-grown cells of Nocardia globerula 432

Nocardia globerula strain 432 was able to synthesize triacylglycerols (TAG) during cultivation on 2,6,10,14-tetramethyl pentadecane (pristane) under nitrogen-limiting conditions. Within these cells, 4,8,12-trimethyl tridecanoic acid was the major fatty acid detected. Fatty acids with an odd number of carbon atoms and minor amounts of even-numbered fatty acids were also observed. Experiments carried out with acrylic acid, an inhibitor of beta-oxidation, suggested that odd-numbered fatty acids such as C15:0, C17:0 and 10-methyl C17:0 were synthesized de novo using propionyl-CoA, the beta-oxidation product, as precursor. Although N. globerula 432 incorporated mainly straight chain fatty acids into TAG, the branched fatty acid 4,8,12-trimethyl tridecanoic acid also appeared, to some extent, in the acylglycerols. The importance of TAG biosynthesis by pristane-grown cells of N. globerula strain 432 is discussed.     

Current progress in the fatty acid metabolism in Cryptosporidium parvum

Cryptosporidium parvum is one of the apicomplexans that can cause severe diarrhea in humans and animals. The slow development of anti-cryptosporidiosis chemotherapy is primarily due to the poor understanding on the basic metabolic pathways in this parasite. Many well-defined or promising drug targets found in other apicomplexans are either absent or highly divergent in C. parvum. The recently discovered apicoplast and its associated Type II fatty acid synthetic enzymes in Plasmodium, Toxoplasma, and Eimeria apicomplexans are absent in C. parvum, suggesting this parasite is unable to synthesize fatty acids de novo. However, C. parvum possesses a giant Type I fatty acid synthase (CpFAS1) that makes very long chain fatty acids using mediate or long chain fatty acids as precursors. Cryptosporidium also contains a Type I polyketide synthase (CpPKS1) that is probably involved in the production of unknown polyketide(s) from a fatty acid precursor. In addition to CpFAS1 and CpPKS1, a number of other enzymes involved in fatty acid metabolism have also been identified. These include a long chain fatty acyl elongase (LCE), a cytosolic acetyl-CoA carboxylase (ACCase), three acyl-CoA synthases (ACS), and an unusual "long-type" acyl-CoA binding protein (ACBP), which allows us to hypothetically reconstruct the highly streamlined fatty acid metabolism in this parasite. However, C. parvum lacks enzymes for the oxidation of fatty acids, indicating that fatty acids are not an energy source for this parasite. Since fatty acids are essential components of all biomembranes, molecular and functional studies on these critical enzymes would not only deepen our understanding on the basic metabolism in the parasites, but also point new directions for the drug discovery against C. parvum and other apicomplexan-based diseases.     

Metabolic Turnover of Fatty Acids and Acylglycerols in Rat Sciatic Nerve

To explain the discrepancy between the low level and high metabolic activity of endoneurial free fatty acids (FFAs) and triacylglycerol (TG), levels of de novo synthesized FFA and acylglycerols were measured in rat sciatic endoneurium at various intervals after endoneurial microinjection of [14C]acetate. Soon after injection (less than 10 min), the [14C]acetate was metabolized to FFA and incorporated into diacylglycerol (DG), TG, sterols, ceramides, and various phospholipids. The proportions of 14C-labeled FFA, DG, TG, and ceramides to total 14C-labeled lipids decreased, whereas those of phospholipids and cerebrosides increased with time after injection. These findings suggest that rapid turnover of FFA and TG may contribute to their low level in sciatic endoneurium. The de novo synthesized fatty acids were largely incorporated into phosphatidylcholine (approximately 50% of total 14C-labeled phospholipids), probably via the cytidine nucleotide pathway using 1,2-DG as a metabolic intermediate. Hydrolysis of [14C]phosphatidylcholine revealed that fatty acids were labeled at both the C-1 (approximately 43%) and C-2 (approximately 57%) positions. On the other hand, a temporal association between decreased amounts of 14C-label in ceramides and increased amounts of 14C-label in sphingomyelin and galactocerebrosides supports the hypothesis that peripheral nerve galactocerebroside is derived, in vivo, from ceramide via acylation of sphingosine. This exclusive labeling of endoneurial lipids by endoneurial microinjection of labeled precursor provides a unique model for studying synthesis and metabolic turnover of membrane lipids in experimental neuropathies.     

Plasma Membrane Sterols Are Essential for Sensing Osmotic Changes in the Halotolerant Alga Dunaliella

The halotolerant alga Dunaliella responds to hyperosmotic stress by synthesis of massive amounts of glycerol. The trigger for this osmotic response is the change in cell volume, but the mechanism that senses volume changes is not known. Preincubation of Dunaliella salina with tridemorph, a specific inhibitor of sterol biosynthesis, inhibits glycerol synthesis and volume recovery. The inhibition is associated with suppression of [14C]bicarbonate incorporation into sterols and is correlated with pronounced depletion of plasma membrane sterols. Incubation of sterol-depleted cells with cholesterol hemisuccinate restores the capacity for volume regulation in response to hyperosmotic stress. Tridemorph as well as lovastatin also inhibit volume changes that are induced by high light in Dunaliella bardawil, a species that responds to high light intensity by synthesis of large amounts of [beta]-carotene. These volume changes result from accumulation of glycerol and are associated with de novo synthesis of sterols. The major plasma membrane sterol in D. salina and the high-light-induced sterol in D. bardawil co-migrate with ergosterol on thin-layer chromatography and on reversed-phase high-performance liquid chromatography. These results suggest that the osmosensory mechanism in Dunaliella resides in the plasma membrane, and that sterols have an important role in sensing osmotic changes.     

Intermediary metabolism of adipose tissue.

Metabolism of ruminant adipocytes involves the synthesis and mobilization of lipids. Rates of lipid synthesis from the uptake of preformed fatty acids (via lipoprotein lipase) and de novo synthesis of fatty acids are related to the energy balance. Acetate is the major carbon source for fatty acid synthesis with NADPH originating from the pentose cycle and the isocitrate cycle. Ruminant adipose tissue lacks the ability to utilize for lipogenesis those substrates that generate mitochondrial acetyl CoA because of an absence of ATP citrate-lyase and NADP-malate dehydrogenase. Lipid mobilization in ruminant adipocytes is apparently regulated via cAMP levels and a summary of the compounds investigated for lipolytic responses is presented. The control of lipid synthesis and mobilization is interrelated in ruminant adipose tissue. The coordinated manner in which these two functions are regulated is examined with regard to adipocyte responses to insulin and epinephrine. In both lipid synthesis and lipid mobilization, ruminant adipocytes are uniquely different from nonruminant adipose tissue. The physiological significance and possible basis for these species differences in adipose metabolism are discussed.     

Early diagenesis of organic matter in lacustrine sediments in terms of methane fermentation

The amounts of organic compounds such as amino acids (proteinaceous substances), fatty acids, and sterols in lacustrine sediments were found to decrease greatly from the mud‐water interface to depths of 20 cm and less at greater core depths. The amounts of methane produced by microbial activity in subsamples incubated for 40 days at 27°C were also found to decrease with core depth, apparently limited by the exhaustion of organic substrates subject to methane fermentation. Decomposition rates of certain organic compounds added to sediment samples were determined by incubation experiments at different temperatures for 40 to 450 days. Experimental results with mixed cultures of microorganisms in lacustrine sediments indicated the following: (a) The marked decomposition of organic compounds such as amino acids, fatty acids, and sterols in the lacustrine sediments resulted mainly in the formation of methane, (b) Unsaturated compounds were more readily decomposed with the ultimate formation of methane than saturated compounds, (c) The susceptibility of saturated fatty acids to methane formation decreased in proportion to their molecular weights.     

30 Seasonal control of phytoplankton biomass and productivity in coastal british columbia lakes and reservoirs

The algal class Chlorarachniophyceae is comprised of a small group of unicellular eukaryotic algae that are often characterized by an unusual amoeboid morphology. This morphology is hypothesized to be the result of a secondary endosymbiosis in which a green alga was engulfed as prey by a nonphotosynthetic amoeba or amoebaflagellate. Whereas much is known about the phylogenetic relationships of individual chlorarachniophytes to one another, and to possible ancestral host organisms in the genera Cercomonas and Heteromita , little is known about their physiology, particularly that of their lipids. In an initial effort to characterize the lipids of this algal class, seven organisms were examined for their fatty acid and sterol composition. These included Bigelowiella natans, Chlorarachnion globusum, Chlorarachnion reptans, Gymnochlora stellata, Lotharella amoeboformis, Lotharella globosa , and Lotharella sp . Fatty acids associated with chloroplast-associated glycolipids, cytoplasmic membrane-associated phospholipids, and storage triglycerides were characterized. Glycolipid fatty acids were found to be of limited composition, containing principally eicosapentaenoic acid [20:5(n-3)] and hexadecanoic acid (16:0), which ranged in relative percentage from 67–90% and 10–29%, respectively, in these seven organisms. Triglyceride-associated fatty acids were found to be similar. Phospholipid fatty acid composition was more variable. The principal phospholipid fatty acids, 16:0 (25–32%) and a compound tentatively identified as docosapentaenoic acid [22:5(n-3)] (26–35%), were found along with a number of C18 and C20 fatty acids. All organisms contained two sterols as free sterols. These were tentatively identified as 24-ethylcholesta-5,22E-dien-3b-ol (stigmasterol; 70–95%) and 24-methylcholesta-5,22E-dien-3b-ol (brassicasterol; 5–30%).