A new prostaglandin, C22-PGF4α, synthesized from docosahexaenoic acid (C22:6n3) by trout gill

The concentrations of prostaglandins PGE₃ and PGF_3α were 214 and 1500 ng/g wet trout gill tissue, respectively. A new prostaglandin, tentatively identified by gas chromatography/mass spectrometry as C22-PGF_4α (590 ng/g wet tissue) was discovered. This was synthesized from docosahexaenoic acid.     

Evaluation of liquid residues from beer and potato processing for the production of docosahexaenoic acid (C22:6n-3, DHA) by native thraustochytrid strains

Liquid residues from beer (RB) and potato (RP) processing were evaluated as carbon sources for the production of docosahexaenoic acid (C22:6n-3, DHA) by two native Thraustochytriidae sp., M12-X1 and C41, in shaking flask experiments. Results were compared with those obtained in the fermentations of glucose, maltose, soluble starch and ethanol. Both strains produced the highest biomass concentration (2.3 g/L) in the fermentation of RB supplemented with nitrogen sources [yeast extract (YE) and monosodium glutamate (MSG)]. DHA content in the fatty acids produced by the native thraustochytrids was dependent on the fermented carbon source; the fatty acids from biomass grown on carbon sources that permitted a lower growth rate contained more DHA. The highest DHA productivity [55.1 mg/(day L)] was obtained in the fermentation of RB-YE-MSG by M12-X1 strain. In this medium, M12-X1 strain grew at a specific growth rate of 0.014 h^?1 and total fatty acid content in the biomass was 41.3%. Production of DHA by M12-X1 strain followed a non-growth rate associated pattern and DHA content in the biomass decreased significantly after growth ceased.     

Regulation of docosahexaenoic acid production by Schizochytrium sp.: effect of nitrogen addition

Docosahexaenoic acid (DHA) percentage in total fatty acids (TFAs) is an important index in DHA microbial production. In this study, the change of DHA percentage in response to fermentation stages and the strategies to increase DHA percentage were investigated. Two kinds of conventional nitrogen sources, monosodium glutamate (MSG) and ammonium sulfate (AS), were tested to regulate DHA synthesis. Results showed that MSG addition could accelerate the substrate consumption rate but inhibit lipid accumulation, while AS addition could increase DHA percentage in TFAs effectively but extend fermentation period slightly. Finally, the AS addition strategy was successfully applied in 7,000-L fermentor and DHA percentage in TFAs and DHA yield reached 46.06 % and 18.48 g/L, which was 19.54 and 17.41 % higher than that of no-addition strategy. This would provide guidance for the large-scale production of the other similar polyunsaturated fatty acid, and give insight into the nitrogen metabolism in oil-producing microorganisms.     

Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp.

The effect of aeration on the performance of docosahexaenoic acid (DHA) production by Schizochytrium sp. was investigated in a 1,500-L bioreactor using fed-batch fermentation. Six parameters, including specific growth rate, specific glucose consumption rate, specific lipid accumulation rate, cell yield coefficient, lipid yield coefficient, and DHA yield coefficient, were used to understand the relationship between aeration and the fermentation characteristics. Based on the information obtained from the parameters, a stepwise aeration control strategy was proposed. The aeration rate was controlled at 0.4 volume of air per volume of liquid per minute (vvm) for the first 24 h, then shifted to 0.6 vvm until 96 h, and then switched back to 0.4 vvm until the end of the fermentation. High cell density (71 g/L), high lipid content (35.75 g/L), and high DHA percentage (48.95%) were achieved by using this strategy, and DHA productivity reached 119 mg/L h, which was 11.21% over the best results obtained by constant aeration rate.     

Formula alpha-linolenic (18:3(n - 3)) and linoleic (18:2(n - 6)) acid influence neonatal piglet liver and brain saturated fatty acids, as well as docosahexaenoic acid (22:6(n - 3)).

Saturated fatty acids can be synthesized de novo and play a role in determining properties of structural membranes. The effect of dietary essential fatty acids, linoleic acid (18:2(n - 6)) and alpha-linolenic acid (18:3(n - 3)), on the saturated fatty acid content of membrane phospholipid has not previously been considered in newborn nutrition. The studies report the effect of low (1% fatty acids) or high (4%) formula 18:3(n - 3) with low (16%) or high (30-35%) formula 18:2(n - 6) on the saturated and unsaturated fatty acid composition of liver and brain structural lipid of piglets fed formula from birth for 15 days. A significant inverse relationship between the formula % 18:3(n - 3), but not 18:2(n - 6), and the liver phospholipid palmitic acid (16:0) was found. This may indicate a possible effect of dietary 18:3(n - 3) on de novo synthesis of 16:0 and requires further investigation. Monounsaturated fatty acids in both liver and brain were significantly lower in response to high 18:3(n - 3) and to high 18:2(n - 6) plus low 18:1(n - 9) in the formula. Liver phospholipid and brain total lipid % docosahexaenoic acid (22:6(n - 3)) were significantly higher when formula containing 4% rather than 1% 18:3(n - 3) was fed, suggesting that 1% 18:3(n - 3) may limit tissue (n - 3) fatty acid accretion. These results suggest that future studies of essential fatty acid requirements, specifically 18:3(n - 3), should consider possible influences on the saturated fatty acids which also play a functional role in tissue structural lipids.     

Formation of 15-lipoxygenase product from docosahexaenoic acid (22:6w3) by human platelets

The metabolism of docosahexaenoic acid (22:6w3) by 15-lipoxygenase activity of washed human platelets was investigated. Platelets produced 17-hydroxydocosahexaenoic acid (HDHE) when incubated with 22:6w3. Similarly, 15-hydroxyeicosatetraenoic acid (HETE) and 13- and 9-hydroxyoctadecadienoic acids (HODD) were produced when incubated with 20:4w6 and 18:2w6, respectively. However, these products were observed only as minor components in the platelet incubation mixture. Control studies with carefully purified platelets and mononuclear cells indicated that these products were formed by the platelets. Chiral phase HPLC analysis indicated that these compounds were mainly in the S configuration with the exception of the 9-HODD, thus, confirming that a lipoxygenase is responsible for their production. The 9-HODD produced by platelets was a racemic mixture.     

Efficient docosahexaenoic acid production by Schizochytrium sp. via a two-phase pH control strategy using ammonia and citric acid as pH regulators

The cell growth and docosahexaenoic acid (DHA) synthesis of Schizochytrium sp. are closely related to the culture pH. A two-phase pH control strategy based on nitrogen consumption was developed in which pH 7.0 was used for biomass accumulation and pH 5.0 for DHA synthesis. Using this strategy, the cell dry weight and DHA content reached 98.07 and 25.85 g/L, respectively. Furthermore, ammonia and citric acid were used as pH regulators. Application of citric acid further resulted in 7.88 and 4.87% improvements of total lipids and the ratio of DHA to total fatty acids, respectively. Ammonia, as a suitable nitrogen source, promoted non-lipid biomass accumulation. Using this method, a maximum DHA yield of 32.75 g/L was obtained with non-lipid biomass (58.01 g/L) and the ratio of DHA to total fatty acids (52.36%). This study provides an easy strategy for large-scale industrial production of DHA via high-cell-density fermentation of Schizochytrium sp.     

Trans-4-hydroxy-2-hexenal is a neurotoxic product of docosahexaenoic (22:6; n-3) acid oxidation

Lipid peroxidation of docosahexaenoic (22:6; n-3) acid (DHA) is elevated in the CNS in patients with Alzheimer's disease and in animal models of seizure and ethanol withdrawal. One product of DHA oxidation is trans -4-hydroxy-2-hexenal (HHE), a six carbon analog of the n-6 fatty acid derived trans -4-hydroxy-2-nonenal (HNE). In this work, we studied the neurotoxic potential of HHE. HHE and HNE were toxic to primary cultures of cerebral cortical neurons with LD₅₀'s of 23 and 18 μmol/L, respectively. Toxicity was prevented by the addition of thiol scavengers. HHE and HNE depleted neuronal GSH content identically with depletion observed with 10 μmol/L of either compound. Using an antibody raised against HHE–protein adducts, we show that HHE modified specific proteins of 75, 50, and 45 kDa in concentration- and time-dependent manners. The time-dependent formation of HHE differed from that of F₄-neuroprostanes following in vitro DHA oxidation likely as a result of the different oxidation pathways involved. Using purified mitochondrial aldehyde dehydrogenase ALDH5A, we found that HHE was oxidized 6.5-fold less efficiently than HNE. Our data demonstrate that HHE and HNE have similarities but also differences in their neurotoxic mechanisms and metabolism.     

Transacylation of 1-O-alkyl-SN-glycero-3-phosphocholine (lyso platelet-activating factor) and 1-O-alkenyl-SN-glycero-3-phosphoethanolamine with docosahexaenoic acid (22:6 omega 3)

[14C]22:6 (docosahexaenoic acid) was rapidly incorporated into cellular lipids in rabbit alveolar macrophages. After removal of free [14C]22:6, the radioactivity in diacyl-glycerophosphocholine (GPC) gradually decreased with a concomitant increase in [14C]22:6 in alkylacyl-GPC and alkenylacyl-glycerophosphoethanolamine (GPE), indicating that [14C]22:6 was transferred from diacyl-GPC to these ether lipid fractions. In fact, macrophage microsomes were shown to catalyze the transfer of [14C]22:6 from exogenously added diacyl-GPC to 1-alkyl-GPC (lyso platelet-activating factor) and 1-alkenyl-GPE. These results are the first evidence for the involvement of the transacylation system in the metabolism of C22 polyunsaturated fatty acids and lyso platelet-activating factor.     

Evidence for peroxisomal retroconversion of adrenic acid (22:4(n-6)) and docosahexaenoic acids (22:6(n-3)) in isolated liver cells

The intracellular localization of the oxidation of [2-14C]adrenic acid (22:4(n-6)) and [1-14C]docosahexaenoic acid (22:6(n-3)) was studied in isolated liver cells. The oxidation of 22:4(n-6) was 2-3-times more rapid than the oxidation of 22:6(n-3), [1-14C]arachidonic acid (20:4(n-6)) or [1-14C]oleic acid (18:1). (+)-Decanoylcarnitine and lactate, both known to inhibit mitochondrial beta-oxidation, reduced the oxidation of 18:1 distinctly more efficiently than with 22:4(n-6) and 22:6(n-3). In liver cells from rats fed a diet containing partially hydrogenated fish oil, the oxidation of 22:6(n-6) and 22:6(n-3) was increased by 30-40% compared with cells from rats fed a standard pellet diet. With 18:1 as substrate, the amount of fatty acid oxidized was very similar in cells from animals fed standard pellets or partially hydrogenated fish oil. Shortened fatty acids were not produced from [5,6,8,9,11,12,14,15-3H]arachidonic acid. In hepatocytes from rats starved and refed 20% fructose, a large fraction of 14C from 22:4 was recovered in 14C-labelled C14-C18 fatty acids. Oxidation of 22:4 thus caused a high specific activity of the extramitochondrial pool of acetyl-CoA. The results suggest that 22:4(n-6) and to some extent 22:6(n-3) are oxidized by peroxisomal beta-oxidation and by this are retroconverted to arachidonic acid and eicosapentaenoic acid.     

Inhibition of neuronal apoptosis by docosahexaenoic acid (22:6n-3). Role of phosphatidylserine in antiapoptotic effect

Enrichment of Neuro 2A cells with docosahexaenoic acid (22:6n-3) decreased apoptotic cell death induced by serum starvation as evidenced by the reduced DNA fragmentation and caspase-3 activity. The protective effect of 22:6n-3 became evident only after at least 24 h of enrichment before serum starvation and was potentiated as a function of the enrichment period. During enrichment 22:6n-3 incorporated into phosphatidylserine (PS) steadily, resulting in a significant increase in the total PS content. Similar treatment with oleic acid (18:1n-9) neither altered PS content nor resulted in protective effect. Hindering PS accumulation by enriching cells in a serine-free medium diminished the protective effect of 22:6n-3. Membrane translocation of Raf-1 was significantly enhanced by 22:6n-3 enrichment in Neuro 2A cells. Consistently, in vitro biomolecular interaction between PS/phosphatidylethanolamine /phosphatidylcholine liposomes, and Raf-1 increased in a PS concentration-dependent manner. Collectively, enrichment of neuronal cells with 22:6n-3 increases the PS content and Raf-1 translocation, down-regulates caspase-3 activity, and prevents apoptotic cell death. Both the antiapoptotic effect of 22:6n-3 and Raf-1 translocation are sensitive to 22:6n-3 enrichment-induced PS accumulation, strongly suggesting that the protective effect of 22:6n-3 may be mediated at least in part through the promoted accumulation of PS in neuronal membranes.     

High levels of docosahexaenoic acid (22:6n-3)-containing phospholipids in high-frequency contraction muscles of hummingbirds and rattlesnakes

Phospholipids containing docosahexaenoic acid (22:6n-3) have been proposed to be required as conformational cofactors for the functional assembly of membrane proteins such as rhodopsin, ion pumps and the various complexes of the mitochondrial electron transport chain (Infante, 1987, Mol. Cell. Biochem. 74, 111-116; Infante and Huszagh, 2000, FEBS Lett. 468, 1-5). This hypothesis predicts that high-frequency contraction muscles, which are endowed with a high content of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and mitochondrial respiration enzymes, would have higher concentrations of 22:6n-3-containing phospholipids when compared with other muscles in the same species known to have a much lower contraction frequency. We have analyzed the fatty acid composition of ruby-throated hummingbird (Archilochus colubris) pectoral and leg muscles and of rattlesnake (Crotalus atrox) shaker and ventral muscles. We have found that hummingbird pectoral muscles, which are high contraction frequency muscles with the highest known respiratory rate among vertebrates, have a 22:6n-3 concentration of 20.8% vs. 4.9% for the low frequency leg muscles. Similarly, rattler muscles in rattlesnakes, also high contraction frequency muscles, have a higher 22:6n-3 concentration than that of their ventral muscles (15.1% vs. 10.6%, respectively). These results are consistent with a specific molecular role for 22:6n-3-containing phospholipids, as proposed.     

Biosynthesis of docosahexaenoic acid (DHA, 22:6-4, 7,10,13,16,19): two distinct pathways

Docosahexaenoic acid (DHA) has long been recognized for its beneficial effect in humans, but its biosynthetic pathway has not been clearly established until recently. According to Sprecher, in mammals, DHA is synthesized via a retro-conversion process in peroxisomes-the aerobic delta4 desaturation-independent pathway. Recent identification of a Thraustochytrium delta4 desaturase indicates that delta4 desaturation is indeed involved in DHA synthesis in Thraustochytrium. More interestingly, an alternative pathway for DHA biosynthesis-the anaerobic polyketide synthase pathway was also reported recently to occur in Schizochytrium, another member of the Thraustochytriidae. This mini-review attempts to assess the latest research on these distinct pathways for DHA biosynthesis.     

Different fermentation strategies by Schizochytrium mangrovei strain pq6 to produce feedstock for exploitation of squalene and omega‐3 fatty acids

Schizochytrium mangrovei strain PQ 6 was investigated for coproduction of docosahexaenoic acid (C22: 6ω‐3, DHA ) and squalene using a 30‐L bioreactor with a working volume of 15 L under various batch and fed‐batch fermentation process regimes. The fed‐batch process was a more efficient cultivation strategy for achieving higher biomass production rich in DHA and squalene. The final biomass, total lipid, unsaponifiable lipid content, and DHA productivity were 105.25 g · L^?1, 43.40% of dry cell weight, 8.58% total lipid, and 61.66 mg · g^?1 · L^?1, respectively, after a 96 h fed‐batch fermentation. The squalene content was highest at 48 h after feeding glucose (98.07 mg · g^?1 of lipid). Differences in lipid accumulation during fermentation were correlated with changes in ultrastructure using transmission electron microscopy and Nile Red staining of cells. The results may be of relevance to industrial‐scale coproduction of DHA and squalene in heterotrophic marine microalgae such as Schizochytrium .     

Enhancement of docosahexaenoic acid production by Schizochytrium sp. using a two-stage oxygen supply control strategy based on oxygen transfer coefficient

Aims: To improve the yield and productivity of docosahexaenoic acid (DHA) by Schizochytrium sp. in terms of the analysis of microbial physiology. Methods and Results: A two‐stage oxygen supply control strategy, aimed at achieving high concentration and high productivity of DHA, was proposed. At the first 40 h, K_La was controlled at 150·1 h^?1 to obtain high μ for cell growth, subsequently K_La was controlled at 88·5 h^?1 to maintain high q_p for high DHA accumulation. Finally, the maximum lipid, DHA content and DHA productivity reached 46·6, 17·7 g l^?1 and 111 mg l^?1 h^?1, which were 43·83%, 63·88% and 32·14% over the best results controlled by constant K_La. Conclusions: This paper described a two‐stage oxygen supply control strategy based on the kinetic analysis for efficient DHA fermentation by Schizochytrium sp. Significance and Impact of the study: This study showed the advantage of two‐stage control strategy in terms of microbial physiology. As K_La is a scaling‐up parameter, the idea developed in this paper could be scaled‐up to industrial process and applied to other industrial biotechnological processes to achieve both high product concentration and high productivity.     

A comparison of the effects of linolenic (18:3 omega 3) and docosahexaenoic (22:6 omega 3) acids on phospholipid bilayers

The class of long chain polyunsaturated fatty acids known as omega-3 are believed to be involved in prevention of a number of human afflictions. The mode of action for two of the most common omega-3 fatty acids, linolenic 18:3 delta 9,12,15 and docosahexaenoic 22:6 delta 4,7,10,13,16,19 (DHA), is not known. One suggestion is that they may be incorporated into membranes and there provide some specific function. Here we compare the effects of DHA and its metabolic precursor linolenic acid on the membrane properties of fluidity, fusion and permeability. The fatty acids were investigated as both free fatty acids and mixed chain 18:0, 18:3 and 18:0, 22:6 phosphatidylcholines (PCs). Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and a series of anthracene stearic acid probes indicates 20 mol% incorporation of either fatty acid into dipalmitoylphosphatidylcholine bilayers broadens and depresses the temperature of the phase transition, but has almost no effect on fluidity in the liquid crystalline state. Similar fluidity was also observed in the liquid crystalline bilayers of the mixed chain PCs using the same set of fluorescent fatty acid probes. In contrast, DHA as a free fatty acid or as part of a mixed chain PC, causes a much greater enhancement than linolenic acid of the rates of fusion and permeability as monitored by fluorescence resonance energy transfer and aqueous compartment mixing (fusion) and by lipid vesicle swelling in isotonic erythritol, (permeability). These experiments establish a clear distinction between the effects of linolenic acid and DHA in membranes.     

Uptake, release and metabolism of docosahexaenoic acid (DHA, c22:6 omega 3) in human platelets and neutrophils

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.     

The pathway from arachidonic to docosapentaenoic acid (20:4n-6 to 22:5n-6) and from eicosapentaenoic to docosahexaenoic acid (20:5n-3 to 22:6n-3) studied in testicular cells from immature rats

The concentration-dependent metabolism of 1-(14)C-labelled precursors of 22:5n-6 and 22:6n-3 was compared in rat testis cells. The amounts of [(14)C]22- and 24-carbon metabolites were measured by HPLC. The conversion of [1-(14)C]20:5n-3 to [3-(14)C]22:6n-3 was more efficient than that of [1-(14)C]20:4n-6 to [3-(14)C]22:5n-6. At low substrate concentration (4 microM) it was 3.4 times more efficient, reduced to 2.3 times at high substrate concentration (40 microM). The conversion of [1-(14)C]22:5n-3 to [1-(14)C]22:6n-3 was 1.7 times more efficient than that of [1-(14)C]22:4n-6 to [1-(14)C]22:5n-6 using a low, but almost equally efficient using a high substrate concentration. When unlabelled 20:5n-3 was added to a cell suspension incubated with [1-(14)C]20:4n-6 or unlabelled 22:5n-3 to a cell suspension incubated with [1-(14)C]22:4n-6, the unlabelled n-3 fatty acids strongly inhibited the conversion of [1-(14)C]20:4n-6 or [1-(14)C]22:4n-6 to [(14)C]22:5n-6. In the reciprocal experiment, unlabelled 20:4n-6 and 22:4n-6 only weakly inhibited the conversion of [1-(14)C]20:5n-3 and [1-(14)C]22:5n-3 to [(14)C]22:6n-3. The results indicate that if both n-6 and n-3 fatty acids are present, the n-3 fatty acids are preferred over the n-6 fatty acids in the elongation from 20- to 22- and from 22- to 24-carbon atom fatty acids. In vivo the demand for 22-carbon fatty acids for spermatogenesis in the rat may exceed the supply of n-3 precursors and thus facilitate the formation of 22:5n-6 from the more abundant n-6 precursors.     

Increased production of docosahexaenoic acid (22: 6 n-3, DHA) in catfish nutritionally stressed by the feeding of oxidized oils and the modulatory effect of dietary α-tocopheryl acetate

Production of hepatic docosahexaenoic acid in juvenile Clarias gariepinus was significantly increased ( P <0.05) by ingestion of rancid diets and this effect was modulated by dietary vitamin E. This has not been described previously in fish. Causal mechanisms are postulated.