Non-methylene-interrupted fatty acids from marine invertebrates: Occurrence,characterization and biological properties

Marine organisms, in particular invertebrates, have proved to be a major source of unique fatty acid (FA) structures originating from unusual biosynthetic pathways. Among them, non-methylene-interrupted (NMI) FA occur in various molluscs in the wide ranges of concentrations (up to 20%), such as the most often encountered 20:2 Δ5,11, 20:2 Δ5,13, 22:2 Δ7,13 or 22:2 Δ7,15. Such NMI FA have also been reported from algae, echinoderms, sponges, tropical rays, and many other invertebrates. The most intriguing marine invertebrates seem to be sponges that commonly contain very long-chain Δ5,9 FA. A third double bond can occur in the NMI FA as reported in some marine organisms, such as 20:3 Δ7,13,16 or 30:3 Δ5,9,23. Lipids of invertebrates from deep-sea hydrothermal and cold-seep vents gave rise to an intense research activity including reports on unprecedented NMI polyunsaturated FA. The bivalve molluscs are able to synthesize de novo the NMI FA but their precise biological interest is presently not well-known, although structural and functional roles in biological membranes have been suggested, in particular a higher resistance to oxidative processes and microbial lipases. Biosynthetic pathways of Δ5,9 FA in sponges were demonstrated up to C₂₆ FA structures and include particular elongation and desaturation steps. Recently, intense research effort has been conducted to investigate the biomedical potential of these unusual FA. Thus, Δ5,9 FA displayed interesting antiplasmodial activity. The most promising FA topoisomerase I inhibitors to date seem to be the long-chain Δ5,9 FA. This inhibitory activity is probably partially responsible for the toxicity displayed by some of the Δ5,9 FA towards cancer cell lines.     

Calmodulins from muscles of marine invertebrates, scallop and sea anemone

Invertebrate calmodulins of the sea anemone and scallop muscle were isolated and their properties were compared with those of vertebrate calmodulins from rabbit muscle and pig brain. The molecular weights estimated by SDS-polyacrylamide gel electrophoresis were similar to the molecular weight (16,500) of the vertebrate calmodulins. Every calmodulin contained 1 mol each of trimethyllysine and histidine, and high contents of acidic amino acids. The marine invertebrate calmodulins contained only one tyrosine in contrast to two tyrosines in the vertebrate ones. As a result, the UV absorption spectra were clearly different. The Ca2+-induced difference UV absorption spectra of the invertebrate calmodulins were indistinguishable from those of the vertebrate ones in spite of the difference in tyrosine contents. In tryptic peptide maps of invertebrate calmodulins, a few spots different from those of vertebrate calmodulins were observed in the basic and acidic peptide regions. The calmodulins of invertebrate muscles and that of rabbit skeletal muscle were almost indistinguishable in terms of the activation profile of rabbit skeletal myosin light chain kinase.     

Regulation of leptin secretion from white adipocytes by free fatty acids

Norepinephrine stimulates lipolysis and concurrently inhibits insulin-stimulated leptin secretion from white adipocytes. To assess whether there is a cause-effect relationship between these two metabolic events, the effects of fatty acids were investigated in isolated rat adipocytes incubated in buffer containing low (0.1%) and high (4%) albumin concentrations. Palmitic acid (1 mM) mimicked the inhibitory effects of norepinephrine (1 microM) on insulin (10 nM)-stimulated leptin secretion, but only at low albumin concentrations. Studies investigating the effects of the chain length of saturated fatty acids [from butyric (C4) to stearic (C18) acids] revealed that only fatty acids with a chain length superior or equal to eight carbons effectively inhibited insulin-stimulated leptin secretion. Long-chain mono- and polyunsaturated fatty acids constitutively present in adipocyte triglyceride stores (oleic, linoleic, gamma-linolenic, palmitoleic, eicosapentanoic, and docosahexanoic acids) also completely suppressed leptin secretion. Saturated and unsaturated fatty acids inhibited insulin-stimulated leptin secretion with the same potency and without any significant effect on basal secretion. On the other hand, inhibitors of mitochondrial fatty acid oxidation (palmoxirate, 2-bromopalmitate, 2-bromocaproate) attenuated the stimulatory effects of insulin on leptin release without reversing the effects of fatty acids or norepinephrine, suggesting that fatty acids do not need to be oxidized by the mitochondria to inhibit leptin release. These results demonstrate that long-chain fatty acids mimic the effects of norepinephrine on leptin secretion and suggest that they may play a regulatory role as messengers between stimulation of lipolysis by norepinephrine and inhibition of leptin secretion.     

Determination of food sources of marine invertebrates from a subtidal sand community using analyses of fatty acids and stable isotopes

The fatty acid compositions and stable isotope ratios of carbon, nitrogen, and sulfur were analyzed in the bivalve mollusks Mactra chinensis, Pandora pulchella, Felaniella usta, and Megangulus zyonoensis, the polychaete Chaetopterus cautus, and the main sources of organic matter in a subtidal sand bottom community in Vostok Bay (Sea of Japan). The fatty acid composition and stable isotope ratios of some bivalves is likely to be indicative of substantial inputs from benthic microalgae and an important role of microbial food chains. Only the filter-feeding polychaete C. cautus showed similarity in these characteristics to suspended particulate organic matter synthesized by phytoplankton. It is suggested that the contribution of benthic microalgae to the diet of a consumer organism, inferred solely from the carbon stable isotope analysis, can be significantly overestimated.     

Accumulation of americium 243 in selected brackish and marine invertebrates

As an initial step in a programme designed to investigate factors which are of importance in affecting the behaviour of actinides towards certain invertebrates found in estuarine and marine environments, laboratory procedures have been developed to study the accumulation of americium in three species: the polychaete wormNereis diversicolor, the brackish-water amphipodGammarus duebeni and the harpacticoid copepodTisbe holothuriae. The species chosen are considered representative of groups having wide ecological importance. It was found that large differences in concentration factors occurred for the same organisms, depending upon aging of the contaminated medium; much higher and more variable values being found when uptake was from freshly contaminated solutions than from those aged up to a week. The interaction of specimens with physico-chemical reactions of americium which appear to take place within the first few days after its introduction into water are considered to be responsible for these differences. Uptake from contaminated water that had been allowed to age in the absence of organisms appears to be unaffected by subsequent conditioning by specimens. Americium concentration factors show a strong tendency to increase with decreasing size of the species, varying from over 1000 forT. holothuriae to about 3 forN. diversicolor. The possibility that the mechanisms regulating the uptake of actinides in different species may depend upon pH is briefly discussed.     

Hydrogen and volatile fatty acids production from marine macroalgae by anaerobic fermentation

Hydrogen and volatile fatty acids (VFAs) were coproduced from marine macroalgae by anaerobic fermentation using a microbial community. The hydrogen and VFAs production were characterized based on inoculum heat-treatment, methanogen inhibitor addition, operating temperature, and in-situ extraction of VFAs. Maximum hydrogen of 179 mL/g-VS and VFAs concentration of 9.8 g/L were produced from 35 g/L of S. japonica within 5 days of anaerobic fermentation. Hydrogen and VFAs yields were well-correlated with carbohydrate content of substrate. Inoculum heat-treatment significantly improved hydrogen production while the VFAs productivity was affected adversely. The addition of methanogen inhibitors also enhanced the hydrogen production, but the effect on VFAs production was dependent on the type of inhibitor used. Low temperature (25°C) was found to be favorable for high hydrogen and VFAs yield, while high temperature (40°C) and programmed-temperature (35 ~ 45°C) were more favorable for hydrogen and VFAs productivity. Clostridium sp. content was found to be the most abundant at 25°C. An extractive fermentation with anion-exchange resin was tested to recover the VFAs and to control the pH during the anaerobic fermentation.     

Accumulation of diacylglycerol induced in platelets by exogenous fatty acids

Free fatty acids added in ethanol to human platelets prelabelled with [${}^{14}\text{C}$]arachidonate induce an accumulation of radioactive diacylglycerol. Unsaturated fatty acids are ten times more potent than palmitate. Ethanol alone does not alter the distribution of radioactivity. Increasing the concentration of arachidonate leads to increased diacylglycerol formation. The fatty acid effect is independent of thrombin, which itself causes a relatively small change in diacylglycerol levels. Neither the labelled triacylglycerol nor the labelled free fatty acid appears to be the source of the diacylglycerol formed which may arise from the activation of phosphatidylinositol phosphodiesterase.     

Inhibition of yeast phosphatidic-acid synthesis by free fatty acids

Particulate preparations obtained from cells of yeast Saccharomyces sake have been shown to possess glycerolphosphate acyltransferase and 1-acylglycerolphosphate acyltransferase activities. Glycerolphosphate acyltransferase exhibits a high specificity for saturated and monoenoic fatty acyl-CoA thioesters. When palmitoyl-CoA is employed as sole acyl group donor, the major lipid product is lysophosphatidic acid. 1-Acylglycerolphosphate acyltransferase of this yeast species has a rather strict specificity for monoenoic fatty acyl-CoA thioesters as acyl donor. These two acyltransferases are strongly inhibited in vitro by low concentrations of free fatty acids. 1-Acylglycerolphosphate acyltransferase is much more susceptible to fatty acid inhibition than glycerolphosphate acyltransferase. The inhibition is dependent not only on the concentration of fatty acid, but also on the length of exposure to fatty acid. Both saturated and unsaturated fatty acids inhibit the acyltransferase activities. The inhibitory effects of fatty acids cannot be ascribed to a nonspecific surfactant action of fatty acids. The present results support the view that free fatty acid serves as a regulator of glycerolipid synthesis.     

Growth inhibition of Monodus subterraneus by free fatty acids

Monodus subterraneus is a microalga, which is known for its high eicosapentaenoic acid (EPA; 20:5omega3) content. To produce EPA commercially, high volumetric productivities of microalgae are required. These high productivities can be reached in flat panel photobioreactors with small optical paths that have to be operated at high cell densities (>10 g/L). However, at these cell densities a reduction of productivity is observed. This growth inhibition is probably caused by growth inhibitors released by the microalgae, which have been suggested to be fatty acids. Our aim was to investigate if free fatty acids produced by M. subterraneus inhibited growth of this species. Therefore a bioassay was developed and saturated, unsaturated and poly-unsaturated fatty acids occurring in Monodus were tested on their growth inhibiting properties. Growth of M. subterraneus was completely inhibited at a saturated concentration (96 microM) of palmitoleic acid (16:1omega7). But, the saturated fatty acid palmitic acid (16:0) and the mono-saturated oleic acid (18:1omega9) were much stronger inhibitors. Growth was inhibited for 50% already at concentrations of 0.4 microM 16:0 and 3 microM 18:1omega9, respectively. These fatty acids probably cause the growth inhibition in high cell density cultures of M. subterraneus.     

Percentage recovery of free fatty acids from bovine muscle lipids by nonchromatographic techniques

The inability of silicic acid to completely separate the neutral lipids from phospholipids has been reported by several investigators (1,2). Hornstein et al. (3) increased the polarity of the solvent system and reported a clean separation of the phospholipid fraction by adsorption on activated silicic acid. Studies on bovine lipids by Hood and Allen (2) utilized acid-washed Florisil to separate the lipid fractions claiming that silicic acid incompletely separates the free fatty acids from the phospholipids. Work performed in this laboratory (4) on bovine lipids confirmed that phospholipids could be effectively separated from free fatty acids by adsorption on silicic acid by incorporating the solvent system described by Hornstein et al. (3). The liquid-liquid partition procedure of Hamilton and McDonald (5) was also found to be sensitive enough to partition the extremely small amount of free fatty acids from the esterified fatty acids. This paper provides evidence for the effectiveness of these methods in separating the frec fatty acids by incorporating an internal standard [1-¹⁴C]palmitic acid.     

Biochemistry of radioiodinated free fatty acids

Summary Radioiodinated free fatty acids have been developed to study myocardial metabolism non-invasively in man. In the present study the distribution of radiolabeled lipids in the myocardium and in arterial and coronary sinus blood was evaluated following injection of three commonly used iodinated fatty acids in fasted (n = 5) and lactate loaded (n = 3) dogs. Five minutes after simultaneous i.v. injection of radioiodinated 17-I-heptadecanoic acid (IHDA),15-(p-I-phenyl) pentadecanoic acid (IPPA) and 15-(p-I-phenyl)-3,3-dimethylpentadecanoic acid (DMIPPA) a biopsy specimen and samples of arterial and coronary sinus blood were taken. After extraction and TLC the relative distribution of radioactivity in the aqueous phase (containing the oxidation products), pellet and organic phase was calculated. The organic phase was further divided into phospholipids, diglycerides, free fatty acids, triglycerides and cholesterolesters. Seventy two percent of IHDA was oxidized, 36% of IPPA and 7% of DMIPPA. The organic phase consisted primarily of triglycerides and phospholipids. The ratios of triglycerides to phospholipids were about the same for IHDA, IPPA and DMIPPA (0.58, 0.65 and 0.50, respectively). Free IHDA in tissue samples was low (4%) and elevated for IPPA and DMIPPA, (17% and 37%). During lactate loading triglycerides were higher for all three fatty acids. For IHDA and IPPA this increase was paralleled by a decrease in the aqueous phase, in case of DMIPPA the aqueous phase remained the same. Five minutes after injection most of the organic phase of both arterial and coronary sinus blood consisted of the injected fatty acids, the aqueous phase contained oxidation products. There were only minor differences during lactate loading. During the evaluation of scintigraphic patterns of the radioiodinated fatty acids under normal conditions (eg at rest) and during elevated lactate levels (eg during exercise) the differences in distribution must therefore be considered.     

Bioluminescent determination of free fatty acids

A simple, highly specific, and sensitive bioluminescent method for determination of free fatty acids in unextracted plasma or serum has been developed. The method is based on the activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3). The pyrophosphate formed is used to phosphorylate fructose 6-phosphate in a reaction catalyzed by the enzyme pyrophosphate-fructose-6-phosphate phosphotransferase (EC 4.1.2.13). The triosephosphates produced from fructose 1,6-bisphosphate by aldolase are oxidized by NAD in the presence of arsenate to 3-phosphoglycerate. The NADH is detected via the bacterial NADH-linked luciferase system. Excellent agreement has been obtained by comparison with accepted methods. In addition, for the determination of serum free fatty acids, the method is particularly applicable for following lipolysis of isolated adipocytes.     

Bacterial production of free fatty acids from freshwater macroalgal cellulose

The predominant strategy for using algae to produce biofuels relies on the overproduction of lipids in microalgae with subsequent conversion to biodiesel (methyl-esters) or green diesel (alkanes). Conditions that both optimize algal growth and lipid accumulation rarely overlap, and differences in growth rates can lead to wild species outcompeting the desired lipid-rich strains. Here, we demonstrate an alternative strategy in which cellulose contained in the cell walls of multicellular algae is used as a feedstock for cultivating biofuel-producing microorganisms. Cellulose was extracted from an environmental sample of Cladophora glomerata-dominated periphyton that was collected from Lake Mendota, WI, USA. The resulting cellulose cake was hydrolyzed by commercial enzymes to release fermentable glucose. The hydrolysis mixture was used to formulate an undefined medium that was able to support the growth, without supplementation, of a free fatty acid (FFA)-overproducing strain of Escherichia coli (Lennen et. al 2010). To maximize free fatty acid production from glucose, an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible vector was constructed to express the Umbellularia californica acyl-acyl carrier protein (ACP) thioesterase. Thioesterase expression was optimized by inducing cultures with 50 μM IPTG. Cell density and FFA titers from cultures grown on algae-based media reached 50% of those (～90 μg/mL FFA) cultures grown on rich Luria-Bertani broth supplemented with 0.2% glucose. In comparison, cultures grown in two media based on AFEX-pretreated corn stover generated tenfold less FFA than cultures grown in algae-based media. This study demonstrates that macroalgal cellulose is a potential carbon source for the production of biofuels or other microbially synthesized compounds.