Production of Chlorella vulgaris as a source of essential fatty acids in a tubular photobioreactor continuously fed with air enriched with CO2 at different concentrations.

To reduce CO2 emissions and simultaneously produce biomass rich in essential fatty acids, Chlorella vulgaris CCAP 211 was continuously grown in a tubular photobioreactor using air alone or air enriched with CO2 as the sole carbon source. While on one hand, nitrogen-limited conditions strongly affected biomass growth, conversely, they almost doubled its lipid fraction. Under these conditions using air enriched with 0, 2, 4, 8, and 16% (v/v) CO2 , the maximum biomass concentration was 1.4, 5.8, 6.6, 6.8, and 6.4 gDB L(-1) on a dry basis, the CO2 consumption rate 62, 380, 391, 433, and 430 mgCO2 L(-1) day(-1) , and the lipid productivity 3.7, 23.7, 24.8, 29.5, and 24.4 mg L(-1) day(-1) , respectively. C. vulgaris was able to grow effectively using CO2 -enriched air, but its chlorophyll a (3.0-3.5 g 100gDB (-1) ), chlorophyll b (2.6-3.0 g 100gDB (-1) ), and lipid contents (10.7-12.0 g 100gDB (-1) ) were not significantly influenced by the presence of CO2 in the air. Most of the fatty acids in C. vulgaris biomass were of the saturated series, mainly myristic, palmitic, and stearic acids, but a portion of no less than 45% consisted of unsaturated fatty acids, and about 80% of these were high added-value essential fatty acids belonging to the ω3 and ω6 series. These results highlight that C. vulgaris biomass could be of great importance for human health when used as food additive or for functional food production.

Identification of genes and pathways involved in the synthesis of Mead acid (20:3n-9), an indicator of essential fatty acid deficiency.

In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n-9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1-6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n-9, 20:1n-9 and 20:2n-9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n-9→(Fads2)→18:2n-9→(Elovl5)→20:2n-9→(Fads1)→20:3n-9 and pathway 2) 18:1n-9→(Elovl5)→20:1n-9→(Fads2)→20:2n-9→(Fads1)→20:3n-9.

TORC1 inhibits GSK3-mediated Elo2 phosphorylation to regulate very long chain fatty acid synthesis and autophagy.

Very long chain fatty acids (VLCFAs) are essential fatty acids with multiple functions, including ceramide synthesis. Although the components of the VLCFA biosynthetic machinery have been elucidated, how their activity is regulated to meet the cell's metabolic demand remains unknown. The goal of this study was to identify mechanisms that regulate the rate of VLCFA synthesis, and we discovered that the fatty acid elongase Elo2 is regulated by phosphorylation. Elo2 phosphorylation is induced upon inhibition of TORC1 and requires GSK3. Expression of nonphosphorylatable Elo2 profoundly alters the ceramide spectrum, reflecting aberrant VLCFA synthesis. Furthermore, VLCFA depletion results in constitutive activation of autophagy, which requires sphingoid base phosphorylation. This constitutive activation of autophagy diminishes cell survival, indicating that VLCFAs serve to dampen the amplitude of autophagy. Together, our data reveal a function for TORC1 and GSK3 in the regulation of VLCFA synthesis that has important implications for autophagy and cell homeostasis.

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation.

Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-γ-linolenic acid (DGLA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbß3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane.

Identification of a Δ5-like fatty acyl desaturase from the cephalopod Octopus vulgaris (Cuvier 1797) involved in the biosynthesis of essential fatty acids.

Long-chain polyunsaturated fatty acids (LC-PUFA) have been identified as essential compounds for common octopus (Octopus vulgaris), but precise dietary requirements have not been determined due, in part, to the inherent difficulties of performing feeding trials on paralarvae. Our objective is to establish the essential fatty acid (EFA) requirements for paralarval stages of the common octopus through characterisation of the enzymes of endogenous LC-PUFA biosynthetic pathways. In this study, we isolated a cDNA with high homology to fatty acyl desaturases (Fad). Functional characterisation in recombinant yeast showed that the octopus Fad exhibited Δ5-desaturation activity towards saturated and polyunsaturated fatty acyl substrates. Thus, it efficiently converted the yeast's endogenous 16:0 and 18:0 to 16:1n-11 and 18:1n-13, respectively, and desaturated exogenously added PUFA substrates 20:4n-3 and 20:3n-6 to 20:5n-3 (EPA) and 20:4n-6 (ARA), respectively. Although the Δ5 Fad enables common octopus to produce EPA and ARA, the low availability of its adequate substrates 20:4n-3 and 20:3n-6, either in the diet or by limited endogenous synthesis from C(18) PUFA, might indicate that EPA and ARA are indeed EFA for this species. Interestingly, the octopus Δ5 Fad can also participate in the biosynthesis of non-methylene-interrupted FA, PUFA that are generally uncommon in vertebrates but have been found previously in marine invertebrates, including molluscs, and now also confirmed to be present in specific tissues of common octopus.

Autistic disorder and phospholipids: A review.

Dysregulated phospholipid metabolism has been proposed as an underlying biological component of neurodevelopmental disorders such as autistic disorder (AD) and attention-deficit/hyperactivity disorder (ADHD). This review provides an overview of fatty acid and phospholipid metabolism and evidence for phospholipid dysregulation with reference to the membrane hypothesis of schizophrenia. While there is evidence that phospholipid metabolism is at least impaired in individuals with AD, it has not been established whether phospholipid metabolism is implicated in causal, mechanistic or epiphenomenological models. More research is needed to ascertain whether breastfeeding, and specifically, the administration of colostrum or an adequate substitute can play a preventative role by supplying the neonate with essential fatty acids (EFAs) at a critical juncture in their development. Regarding treatment, further clinical trials of EFA supplementation are essential to determine the efficacy of EFAs in reducing AD symptomatology and whether supplementation can serve as a cost-effective and readily available intervention.

Extremely limited synthesis of long chain polyunsaturates in adults: implications for their dietary essentiality and use as supplements.

There is considerable interest in the potential impact of several polyunsaturated fatty acids (PUFAs) in mitigating the significant morbidity and mortality caused by degenerative diseases of the cardiovascular system and brain. Despite this interest, confusion surrounds the extent of conversion in humans of the parent PUFA, linoleic acid or alpha-linolenic acid (ALA), to their respective long-chain PUFA products. As a result, there is uncertainty about the potential benefits of ALA versus eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA). Some of the confusion arises because although mammals have the necessary enzymes to make the long-chain PUFA from the parent PUFA, in vivo studies in humans show that asymptotically equal to 5% of ALA is converted to EPA and <0.5% of ALA is converted to DHA. Because the capacity of this pathway is very low in healthy, nonvegetarian humans, even large amounts of dietary ALA have a negligible effect on plasma DHA, an effect paralleled in the omega6 PUFA by a negligible effect of dietary linoleic acid on plasma arachidonic acid. Despite this inefficient conversion, there are potential roles in human health for ALA and EPA that could be independent of their metabolism to DHA through the desaturation - chain elongation pathway.

Metabolic pathway that produces essential fatty acids from polymethylene-interrupted polyunsaturated fatty acids in animal cells.

Sciadonic acid (20:3 Delta-5,11,14) and juniperonic acid (20:4 Delta-5,11,14,17) are polyunsaturated fatty acids (PUFAs) that lack the Delta-8 double bond of arachidonic acid (20:4 Delta-5,8,11,14) and eicosapentaenoic acid (20:5 Delta-5,8,11,14,17), respectively. Here, we demonstrate that these conifer oil-derived PUFAs are metabolized to essential fatty acids in animal cells. When Swiss 3T3 cells were cultured with sciadonic acid, linoleic acid (18:2 Delta-9,12) accumulated in the cells to an extent dependent on the concentration of sciadonic acid. At the same time, a small amount of 16:2 Delta-7,10 appeared in the cellular lipids. Both 16:2 Delta-7,10 and linoleic acid accumulated in sciadonic acid-supplemented CHO cells, but not in peroxisome-deficient CHO cells. We confirmed that 16:2 Delta-7,10 was effectively elongated to linoleic acid in rat liver microsomes. These results indicate that sciadonic acid was partially degraded to 16:2 Delta-7,10 by two cycles of beta-oxidation in peroxisomes, then elongated to linoleic acid in microsomes. Supplementation of Swiss 3T3 cells with juniperonic acid, an n-3 analogue of sciadonic acid, induced accumulation of alpha-linolenic acid (18:3 Delta-9,12,15) in cellular lipids, suggesting that juniperonic acid was metabolized in a similar manner to sciadonic acid. This PUFA remodeling is thought to be a process that converts unsuitable fatty acids into essential fatty acids required by animals.

Biological significance of essential fatty acids.

Essential fatty acids (EFAs)--linoleic acid (LA) and alpha-linolenic acid (ALA) are critical for human survival. EFAs are readily available in the diet. But, to derive their full benefit, EFAs need to be metabolized to their respective long-chain metabolites. EFAs not only form precursors to respective prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs), but also give rise to lipoxins (LXs), resolvins, isoprostanes, and hydroxy- and hydroperoxyeicosatetraenoates. Certain PGs, TXs, and LTs have pro-inflammatory actions whereas LXs and resolvins are anti-inflammatory in nature. Furthermore, EFAs and their long-chain metabolites modulate the activities of angiotensin converting and HMG-CoA reductase enzymes, enhance acetylcholine levels in the brain, increase the synthesis of endothelial nitric oxide, augment diuresis, and enhance insulin action. Thus, EFAs and their metabolites may function as endogenous ACE and HMG-CoA reductase inhibitors, nitric oxide enhancers, beta-blockers, diuretics, anti-hypertensive, and anti-atherosclerotic molecules. In addition, EFAs and their long-chain metabolites react with nitric oxide (NO) to yield respective nitroalkene derivatives that exert cell-signaling actions via ligation and activation of peroxisome proliferator-activated receptors (PPARs). Thus, EFAs and their derivatives have varied biological actions that may have relevance to their involvement in several physiological and pathological processes.

Genetic modification of essential fatty acids biosynthesis in Hansenula polymorpha.

The Delta(6)-desaturase gene isoform II involved in the formation of gamma-linolenic acid (GLA) was identified from Mucor rouxii. To study the possibility of alteration of the synthetic pathway of essential fatty acids in the methylotrophic yeast, Hansenula polymorpha, the cloned gene of M. rouxii under the control of the methanol oxidase (MOX) promoter of H. polymorpha, was used for genetic modification of this yeast. Changes in flux through the n-3 and n-6 pathways in the transgenic yeast were observed. The proportion of GLA varied dramatically depending on the growth temperature and media composition. This can be explained by the effects of either substrate availability or enzymatic activity. In addition to the potential application for manipulating the fatty acid profile, this study provides an attractive model system of H. polymorpha for investigating the deviation of fatty acid metabolism in eukaryotes.

Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants.

We report the production of two very long chain polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), in substantial quantities in a higher plant. This was achieved using genes encoding enzymes participating in the omega3/6 Delta8 -desaturation biosynthetic pathways for the formation of C20 polyunsaturated fatty acids. Arabidopsis thaliana was transformed sequentially with genes encoding a Delta9 -specific elongating activity from Isochrysis galbana, a Delta8 -desaturase from Euglena gracilis and a Delta5 -desaturase from Mortierella alpina. Instrumental in the successful reconstitution of these C20 polyunsaturated fatty acid biosynthetic pathways was the I. galbana C18-Delta9 -elongating activity, which may bypass rate-limiting steps present in the conventional Delta6 -desaturase/elongase pathways. The accumulation of EPA and AA in transgenic plants is a breakthrough in the search for alternative sustainable sources of fish oils.

Arachidonic acid synthetic pathways of the oyster protozoan parasite, Perkinsus marinus: evidence for usage of a delta-8 pathway.

The meront stage of the oyster protozoan parasite, Perkinsus marinus, is capable of synthesizing saturated and unsaturated fatty acids including the essential fatty acid, arachidonic acid [20:4(n-6)]. Eukaryotes employ either delta-6 (Delta-6) or delta-8 (Delta-8) desaturase pathway or both to synthesize arachidonic acid. To elucidate the arachidonic acid synthetic pathways in P. marinus, meronts were incubated with deuterium-labeled precursors [18:1(n-9)-d6, 18:2(n-6)-d4, 18:3(n-3)-d4, and 20:3(n-3)-d8]. The lipids were extracted, converted to fatty acid methyl esters, and analyzed using gas chromatography/mass spectrometry and gas chromatography/flame ionization detection. Deuterium-labeled 18:2(n-6), 20:2(n-6), 20:3(n-6), and 20:4(n-6) were detected in meront lipids after 1-, 3-, 5-, and 10-day incubation with 18:1(n-9)-d6. Deuterium-labeled 20:2(n-6), 20:3(n-6) and 20:4(n-6) were found in lipids from meronts after incubation with 18:2(n-6)-d4 methyl ester. No labeled 18:3(n-6) was detected in either incubation. Apparently, when incubated with 18:1(n-9)-d6, the parasite first desaturated 18:1(n-9)-d6 to 18:2(n-6)-d6 by Delta-12 desaturase, then to 20:2(n-6)-d6 by elongation, and ultimately desaturated to 20:3(n-6)-d6 and 20:4(n-6)-d6 using the sequential Delta-8 and Delta-5 desaturation. Similarly, when incubated with 18:2(n-6)-d4, P. marinus converted the 18:2(n-6)-d4 to 20:2(n-6)-d4 by elongation and 20:2(n-6)-d4 to 20:3(n-6)-d4 by Delta-8 desaturase then by Delta-5 desaturase to 20:4(n-6)-d4. These results provide evidence that P. marinus employed the Delta-8 rather Delta-6 pathway for arachidonic acid synthesis. Additional support for the presence of a Delta-8 pathway was the demonstrated ability of the parasite to metabolize 18:3(n-3)-d4 to 20:3(n-3)-d4 and 20:4(n-3)-d4, and 20:3(n-3)-d8 to 20:4(n-3)-d6 and 20:5(n-3)-d6 using the sequential position-specific Delta-8 and Delta-5 desaturases.

[Fatty acids in the species of several zygomycete taxa]. / Zhirnye kisloty predstavitelei riada taksonov zigomitsetov.

The composition of fatty acids synthesized de novo by thirty strains of zygomycetes from various taxa was studied. The qualitative fatty acid compositions of the fungal lipids were found to be virtually identical, but there were significant differences in the contents of individual acids. Highly active producers of essential C18 fatty acids, with their content exceeding 30-40% of total fatty acids, were discovered among the fungi of the families Mucoraceae, Pilobolaceae, and Radiomycetaceae. Linoleic acid was found to predominate in the fungi of the genera Radiomyces, Mycotypha, and Circinella, and linolenic acid (identified as its gamma-isomer by gas-liquid chromatography), in the fungi of the genera Absidia, Circinella, Pilaira, and Hesseltinella. The total yield (mg/l) of bioactive acids (C18:3, C18:2, C18:1) varied from 761.4 in Pilaira anomala to 3477.9 in Syncephalastrum racemosum; the total yield of essential acids, from 520.7 in Pilaira anomala to 1154.5 in Hesseltinella vesiculosa; of linoleic acid, from 279.7 in Pilaira anomala to 836.3 in Mycotypha indica; and of linolenic acid, from 120.8 in Mycotypha indica to 708.0 in Hesseltinella vesiculosa. The data on the efficient synthesis of these acids make the actively producing strains promising for biotechnological synthesis of commercially valuable lipids. Linderina pennispora VKM F-1219, a zygomycete of the family Kickxellaceae, which was earlier singled out into the order Kickxellales [12], was shown to differ from zygomycetes of the order Mucorales in having a high content of cis-9-hexadecenoic (palmitoleic) acid, reaching 37.0% of the fatty acid total.

Studies on the metabolism of essential fatty acids in isolated human testicular cells.

The essential fatty acid 22:6(n-3) is a minor component of the Western diet, but a major fatty acid in human testis and semen. In mature spermatozoa, the physical and fusogenic properties of the plasma membrane are probably influenced by its particular fatty acid composition. In this study, the synthesis of 22:6(n-3) and 22:5(n-6) was investigated in isolated human testicular cells. [1-(14)C]20:4(n-6), [1-(14)C]20:5(n-3), [1-(14)C]22:4(n-6) and [1-(14)C]22:5(n-3) were incubated in a 'crude' cell suspension (consisting of a mixture of the cells in the seminiferous tubule), and in fractionated pachytene spermatocytes and round spermatids. The esterification of fatty acids in lipid and phospholipid classes and the fatty acid chain elongation and desaturation were measured. The crude cell suspension metabolized the fatty acids more actively than did the fractionated germ cell suspension, indicating that types of cell other than the germ cells are important for fatty acid elongation and desaturation and thus the production of 22:6(n-3). This finding is in agreement with previous results in rats that indicated that the Sertoli cells are the most important type of cell for the metabolism of essential fatty acids in the testis. Some [1-(14)C]20:5(n-3) was elongated to [(14)C]22:5(n-3) in the fractionated germ cells, but very little was elongated further to [(14)C]24:5(n-3),possibly restricting the formation of [(14)C]22:6(n-3). In the fractionated germ cells, the fatty acid substrates were recovered primarily in the phospholipid fraction, indicating an incorporation in the membranes, whereas in the crude cells, more substrates were esterified in the triacylglycerol fraction. In the phospholipids, more radioactivity was recovered in phosphatidylcholine than in phosphatidylethanolamine and more radioactivity was recovered in phosphatidylethanolamine than in phosphatidylinositol or phosphatidylserine.

Fatty acids in cystic fibrosis.

Cystic fibrosis (CF) is associated with deficiencies in certain essential fatty acids. These deficiencies have been studied in plasma, red blood cells, and mucus and were previously thought to be a result of malnutrition or malabsorption. More recent studies have indicated that these deficiencies are independent of nutritional status. However, these studies examined fatty acids in plasma but not in CF-regulated tissues. In the pancreas, lungs, and ileum of CF knock-out mice, membrane-bound arachidonic acid levels have been shown to be increased while docosahexaenoic acid levels are decreased. This lipid abnormality is reversed following oral administration of docosahexaenoic acid (DHA). In addition, DHA therapy reverses the increased neutrophil infiltration in the lungs of CF knock-out mice. Further studies are required to determine the mechanism by which CF gene mutations lead to this lipid abnormality.

[Diet as a determinant of central nervous system development: role of essential fatty acids]. / La dieta como determinante del desarrollo del sistema nervioso central: rol de los ácidos grasos esenciales.

Nervous system is second to adipose tissue in containing the highest lipid concentration. Membrane phospholipids possess a high content of long chain polyunsaturated fatty acids (LCPUFA) of the n-3 and n-6 families, derived from the corresponding essential fatty acids. Docosahexaenoic acid (22:6n-3) is found in the highest concentrations in the gray matter and the photoreceptors of the retina. n-3 LCPUFA deficiency in infants, mainly if born preterm, modifies neural functions causing learning disabilities and visual function abnormalities. The adequate lipid nutrition of the mother before and during pregnancy and in breast feeding determine the lipid transfer of fatty acids to the fetus and infant, respectively. LCPUFA are conditionally essential in preterm infants, born with lower lipid depots. The formulation of infant formulas, mainly for preterm babies, should include adequate proportions of n-3 and n-6 LCPUFA.

La dieta como determinante del desarrollo del sistema nervioso central: rol de los ácidos grasos asenciales / Diet as a determinant of the central nervous system development: role of essential fatty acids

El sistema nervioso central es el segundo tejido más rico en lípidos del organismo, luego del adiposo. Los fosfolípidos constituyentes de las membranas cerebrales poseen un alto contenido de ácidos grasos poliisaturados de cadena larga (AGPICL) de las familias n-3 y n-6, derivados de los respectivos ácidos grasos esenciales. Las mayores concentraciones de AGPICL en tejido nervioso corresponden al ácido docosahexaenoico (22:6n-3), en membranas de materia gris de corteza cerebral y fotoreceptores de retina. La deficiencia de AGPICL n-3 en recién nacidos, especialmente de pretérmino, modifica la función nerviosa, provocando dificuldades de aprendizaje y alteraciones de la función visual. El período de mayor acumulación de estos lípidos en cerebro es perinatal. Por esta razón, es importante la alimentación adecuada de la embarazada, antes y durante la gestación, y de la mujer que amamanta, que transfieren los AGPICL al feto y recién nacido, respectivamente. En niños de pretérmino, que nacen con menores reservas de AGPICL, estos tienen la calidad de condicionalmente esenciales. La formulación de las preparaciones destinadas a la alimentación de lactantes, especialmente si son prematuros, debe incluir AGPICL n-3 y n-6 en proporciones adecuadas.

Fish lipids: more than n-3 fatty acids?

Recent studies have shown that marine oils rich in long-chain (C20 and C22) fatty acids (i.e. certain natural marine oils and partially hydrogenated fish oil) may affect the haemostatic balance in a favourable way with regard to coronary heart disease. Such fats have also been found to increase the content of EPA (eicosapentaenoic acid = C20:5 n-3) and to decrease the content of arachidonic acid (C20:4 n-6) in blood lipids, thus affecting the ratio of C20:4 n-6 to C20:5 n-3 in a favourable way with regard to eicosanoid production. It is therefore likely that the positive effects of long-chain monoenoic fatty acids on haemostasis are due to increased synthesis of long-chain essential n-3 fatty acids. According to recent theories the final steps in the synthesis of long-chain essential fatty acids occur in the peroxisomes, which also have a controlling function in essential fatty acid synthesis. Long-chain monoenoic fatty acids are known to enhance peroxisomal beta-oxidation. An hypothesis is therefore advanced that marine oils rich in long-chain monoenoic fatty acids improve haemostasis in a favourable way with regard to coronary heart disease through increased peroxisomal beta-oxidation and increased synthesis of long-chain n-3 fatty acids.