Production of arachidonic acid by Mortierella alpina using wastes from potato chips industry.

AIMS: This research aimed to determine the potential use of wastes from the potato chips industry as a carbon source to develop an economical culture medium for the production of biomass, lipids and arachidonic acid (ARA) by Mortierella alpina. METHODS AND RESULTS: A synthetic culture medium was optimized using a Plackett-Burman and central composite rotatable design, and used as a base to evaluate and characterize the potential use of wastes from the potato chips industry as carbon sources for the production of biomass, lipids and ARA by M. alpina. The waste was selected among other solid and liquid hydrolysed residues/by-products, and local low-cost alternatives for nitrogen sources were also evaluated. After 6 days of fermentation, the biomass concentration reached 20 g l-1 with 40% of total lipids, and a 35% ARA content in the lipids fraction. Savings in production were calculated using a sensitivity analysis for the alternative culture medium in different scenarios. CONCLUSIONS: This study showed a 7% savings in culture media expenses in the production of ARA-enriched biomass of M. alpina, compared to the conventional synthetic culture medium, when waste from the potato chips industry was used as an alternative source of carbon and macro/microelements, supplemented with a low-cost yeast extract alternative. SIGNIFICANCE AND IMPACT OF THE STUDY: The demonstration of the use of potato chips wastes as a low-cost carbon source for the biomass, lipids and ARA production, suggesting an eco-friendly alternative for the use of agri-food wastes for valuable metabolites production.

Improved Production of Arachidonic Acid by Combined Pathway Engineering and Synthetic Enzyme Fusion in Yarrowia lipolytica.

Arachidonic acid (ARA, C20:4) is a typical ω-6 polyunsaturated fatty acid with special functions. Using Yarrowia lipolytica as an unconventional chassis, we previously showed the performance of the Δ-6 pathway in ARA production. However, a significant increase in the Δ-9 pathway has rarely been reported. Herein, the Δ-9 pathway from Isochrysis galbana was constructed via pathway engineering, allowing us to synthesize ARA at 91.5 mg L-1. To further improve the ARA titer, novel enzyme fusions of Δ-9 elongase and Δ-8 desaturase were redesigned in special combinations containing different linkers. Finally, with the integrated pathway engineering and synthetic enzyme fusion, a 29% increase in the ARA titer, up to 118.1 mg/L, was achieved using the reconstructed strain RH-4 that harbors the rigid linker (GGGGS). The results show that the combined pathway and protein engineering can significantly facilitate applications of Y. lipolytica.

Effects of nitrogen and phosphorous stress on the formation of high value LC-PUFAs in Porphyridium cruentum.

This study systematically examined the effect of nitrogen and phosphorous stress on the formation of linoleic acid (LA), arachidonic acid (ARA), and eicosapentaenoic acid (EPA) in Porphyridium cruentum gy-h56. P. cruentum was cultivated in six different media conferring different conditions of nitrogen (N) sufficiency/deprivation and phosphorous (P) sufficiency/limitation/deprivation. Over a 16-day cultivation process, the dry-weight content, proportion of total fatty acids (TFAs), and the concentration in the medium of linoleic acid (LA) were greatly improved by a maximum of 2.5-, 1.6-, and 1.1-fold, respectively, under conditions of N or P deprivation compared with N and P sufficiency. In contrast, levels of EPA or ARA were not enhanced under N or P stress conditions. Additionally, the results showed that N deprivation weakened the impact of P deficiency on the content and proportions of LA and EPA, while P deprivation enhanced the impact of N starvation on the content and proportions of LA and EPA. The conditions of N sufficiency and P deprivation (N+P-) were the optimal conditions for the production of LA, while the optimal conditions for EPA, ARA, and TFAs production were N sufficiency and P limitation (N+P-lim). This study suggests the potential application of combining N removal from saline wastewater with the production of LA, ARA, EPA, and biodiesel.

Different effects of γ-linolenic acid (GLA) supplementation on plasma and red blood cell phospholipid fatty acid composition and calcium oxalate kidney stone risk factors in healthy subjects from two race groups with different risk profiles pose questions about the GLA-arachidonic acid-oxaluria metabolic pathway: pilot study.

Fatty acid (FA) composition of phospholipids in plasma and red blood cells (RBC) can influence calciuria, oxaluria and renal stone formation. In this regard, the ratio of arachidonic acid (AA) and its precursor linoleic acid (LA) appears to be important. Administration of γ-linolenic acid (GLA) has been shown to increase the concentration of dihomo-gamma linoleic acid (DGLA) relative to AA indicating that it may attenuate biosynthesis of the latter. Such effects have not been investigated in race groups having difference stone occurrence rates. Black (B) and white (W) healthy males ingested capsules containing linoleic acid (LA) and GLA, for 30 days. Plasma and RBC total phospholipid (TPL) FA profiles, serum and 24 h urine biomarkers of hypercalciuria and urinary stone risk factors were determined on days 0 and 30. Data were tested for statistical significance using GraphPadInstat version 3.02. Concentration and percentage content of DGLA in plasma TPL increased in W but not in B. Arachidonic acid (AA) did not change in either group. There was no change in calcium excretion in either group but oxalate and citrate excretion increased in W. We suggest that elongation of GLA to DGLA may occur more rapidly than desaturation of DGLA to AA in W and that depressed activity of the enzyme elongase may occur in B. Calciuric and citraturic effects may be dependent on the quantity of LA or on the mass ratio of LA/GLA in the FA supplement. Questions about the mooted DGLA-AA-oxaluria pathway arise. We speculate that there exists a potential for using GLA as a conservative treatment for hypocitraturia. The observation of different responses in B and W indicates that such differences may play a role in stone formation and prevention.

Effects of Mucuna pruriens on Free Fatty Acid Levels and Histopathological Changes in the Brains of Rats Fed a High Fructose Diet.

OBJECTIVE: To investigate free fatty acid levels and histopathological changes in the brain of rats fed a high fructose diet (HFrD) and to evaluate the effects of Mucuna pruriens, known to have antidiabetic activity, on these changes. MATERIALS AND METHODS: The study comprised 28 mature female Wistar rats. The rats were divided into 4 groups, each included 7 rats. Group 1: control; group 2: fed an HFrD; group 3: fed normal rat chow and M. pruriens; group 4: fed an HFrD and M. pruriens for 6 weeks. At the end of 6 weeks, the rats were decapitated, blood and brain tissues were obtained. Serum glucose and triglyceride levels were measured. Free fatty acid levels were measured in 1 cerebral hemisphere of each rat and histopathological changes in the other. The Mann-Whitney U test was used to compare quantitative continuous data between 2 independent groups, and the Kruskal-Wallis test was used to compare quantitative continuous data between more than 2 independent groups. RESULTS: Arachidonic acid and docosahexaenoic acid levels were significantly higher in group 2 than in group 1 (p < 0.05). Free arachidonic acid and docosahexaenoic acid levels in group 4 were significantly less than in group 2 (p < 0.05). Histopathological examination of group 2 revealed extensive gliosis, neuronal hydropic degeneration, and edema. In group 4, gliosis was much lighter than in group 2, and edema was not observed. Neuronal structures in group 4 were similar to those in group 1. CONCLUSIONS: The HFrD increased the levels of free arachidonic acid and docosahexaenoic acid probably due to membrane degradation resulting from possible oxidative stress and inflammation in the brain. The HFrD also caused extensive gliosis, neuronal hydropic degeneration, and edema. Hence, M. pruriens could have therapeutic effects on free fatty acid metabolism and local inflammatory responses in the brains of rats fed an HFrD.

Group X Secreted Phospholipase A2 Releases ω3 Polyunsaturated Fatty Acids, Suppresses Colitis, and Promotes Sperm Fertility.

Within the secreted phospholipase A2(sPLA2) family, group X sPLA2(sPLA2-X) has the highest capacity to hydrolyze cellular membranes and has long been thought to promote inflammation by releasing arachidonic acid, a precursor of pro-inflammatory eicosanoids. Unexpectedly, we found that transgenic mice globally overexpressing human sPLA2-X (PLA2G10-Tg) displayed striking immunosuppressive and lean phenotypes with lymphopenia and increased M2-like macrophages, accompanied by marked elevation of free ω3 polyunsaturated fatty acids (PUFAs) and their metabolites. Studies usingPla2g10-deficient mice revealed that endogenous sPLA2-X, which is highly expressed in the colon epithelium and spermatozoa, mobilized ω3 PUFAs or their metabolites to protect against dextran sulfate-induced colitis and to promote fertilization, respectively. In colitis, sPLA2-X deficiency increased colorectal expression of Th17 cytokines, and ω3 PUFAs attenuated their production by lamina propria cells partly through the fatty acid receptor GPR120. In comparison, cytosolic phospholipase A2(cPLA2α) protects from colitis by mobilizing ω6 arachidonic acid metabolites, including prostaglandin E2 Thus, our results underscore a previously unrecognized role of sPLA2-X as an ω3 PUFA mobilizerin vivo, segregated mobilization of ω3 and ω6 PUFA metabolites by sPLA2-X and cPLA2α, respectively, in protection against colitis, and the novel role of a particular sPLA2-X-driven PUFA in fertilization.

Heating of vegetable oils influences the activity of enzymes participating in arachidonic acid formation in Wistar rats.

Dietary intake of lipids and their fatty acids profile influence many aspects of health. Thermal processing changes the properties of edible oils and can also modify their metabolism, for example, eicosanoids formation. The aim of our study was to verify whether the activity of desaturases can be modified by lipids intake, especially by the fatty acids content. The experimental diets contained rapeseed oil, sunflower oil, and olive oil, both unheated and heated (for 10 minutes at 200 °C each time before administration), and influenced the fatty acids composition in serum and the activity of enzymes participating in arachidonic acid (AA) formation. The activity of desaturases was determined by measuring the amounts of AA formed in vitro derived from linoleic acid as determined in liver microsomes of Wistar rats. In addition, the indices of ∆(6)-desaturase (D6D) and ∆(5)-desaturase (D5D) have been determined. To realize this aim, the method of high-performance liquid chromatography has been used with ultraviolet-visible spectrophotometry detection. Diet supplementation with the oils rich in polyunsaturated fatty acids affects the fatty acids profile in blood serum and the activity of D6D and ∆(5)-desaturase in rat liver microsomes, the above activities being dependent on the kind of oil applied. Diet supplementation with heated oils has been found to increase the amount of AA produced in hepatic microsomes; and in the case of rapeseed oil and sunflower oil, it has also increased D6D activity.

Enhanced arachidonic acid production from Mortierella alpina combining atmospheric and room temperature plasma (ARTP) and diethyl sulfate treatments.

To obtain mutant strains with higher arachidonic acid (ARA) yields, the oleaginous fungus Mortierella alpina was mutated using atmospheric and room temperature plasma (ARTP) coupled with diethyl sulfate (DES). A visual compound filter operation was used in which a screening medium was supplemented with cerulenin, an inhibitor of fatty acid synthase (FAS), and triphenyltetrazolium chloride (TTC). The mutant strain D20 with an ARA production of 5.09 g/L, a 40.61% increase over the original strain (3.62 g/L), was isolated. The relative ARA content increased from 38.99% to 45.64% of total fatty acids. After optimizing fermentation conditions, the maximum ARA yield (6.82 g/L) for strain D20 was obtained in shake flasks. This work provides an appropriate strategy for obtaining high ARA-yield strains by conventional random mutation methods with an efficient screening assay.

Arachidonic acid production by Mortierella alpina using raw crop materials.

BACKGROUND: Arachidonic acid (ARA) is one of the three essential fatty acids, and it is important for human body to keep healthy and is widely used. At present, expensive materials such as glucose and yeast extract are generally reported to be optimal for ARA production. A new cost-effective fermentation process including cheaper material for ARA production is of great significance. METHODS: Feasibility of using corn meal and powdered soybean for fungal growth and lipid accumulation was evaluated by means of single factor test. N-hexadecane concentration was optimized, and the effect of temperature on biomass and ARA content was examined. RESULTS: Mortierella alpina made better use of the aforementioned material as carbon and nitrogen sources for both hyphae growth and ARA production compared with glucose and yeast extract. Maximal levels of 10.9 g/L ARA and 26.1 g/L total lipids were obtained when 66 g/L corn meal, 54 g/L soybean meal and 6% (v/v) n-hexadecane were supplemented. A temperature-shift strategy involved three steps, namely, 30°C (3 days) - 25°C (4 days) - 20°C (4 days), which further improved ARA production by 24.7%. CONCLUSIONS: Several factors such as carbon and nitrogen sources, temperature and dissolved oxygen had great influence on biomass and microbial oil production. Mortierella alpina preferred corn and soybean meal compared with glucose and yeast extract, which would surely alleviate the high cost of ARA production. Based on this study, the new process is both low cost and practicable.

Preventive effects of ZPDC glycoprotein (24 kDa) on hepatotoxicity induced by mercury chloride in vitro and in vivo.

Mercury is a potent environmental contaminant that exerts toxic effect on various vital organs in the human body. Recently, we isolated glycoprotein from Zanthoxylum piperitum DC (ZPDC), which has antioxidant and anticancer effects. In the present study, we determined the preventive effects of ZPDC glycoprotein on hepatic damage induced by mercury chloride (HgCl2 ). We evaluated the activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)], extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), cyclo-oxygenase (COX-2), inducible nitric oxide synthetase (iNOS), and activator protein (AP-1) and the quantitative expressions of nuclear factor E2-related factor (Nrf2), heme oxygenase (HO-1), metallothionein (MT) and reduced glutathione (GSH) in mercury-chloride-exposed (50 µM and 10 mg/kg body weight) primary cultured hepatocytes and ICR mice, using biochemical assays, radioactivity and immunoblot analysis. The results demonstrated that ZPDC glycoprotein decreased the levels of LDH, ALT, HO-1 and MT, whereas it increased the activities of hepatic antioxidant enzymes (SOD, CAT and GPx) and reduced GSH in mercury-chloride-exposed primary cultured hepatocytes. Also, it suppressed arachidonic acid release and expression of ERK, p38 MAPK, COX-2, iNOS, AP-1 and Nrf-2 in primary cultured hepatocytes and ICR mice exposed to mercury chloride. Collectively, ZPDC glycoprotein may have potential applications to prevent hepatotoxicity induced by mercury chloride.

Retroconversion of docosapentaenoic acid (n-6): an alternative pathway for biosynthesis of arachidonic acid in Daphnia magna.

The aim of this study was to assess metabolic pathways for arachidonic acid (20:4n-6) biosynthesis in Daphnia magna. Neonates of D. magna were maintained on [(13)C] enriched Scenedesmus obliquus and supplemented with liposomes that contained separate treatments of unlabeled docosapentaenoic acid (22:5n-6), 20:4n-6, linoleic acid (18:2n-6) or oleic acid (18:1n-9). Daphnia in the control treatment, without any supplementary fatty acids (FA) containing only trace amounts of 20:4n-6 (~0.3% of all FA). As expected, the highest proportion of 20:4n-6 (~6.3%) was detected in Daphnia that received liposomes supplemented with this FA. Higher availability of 18:2n-6 in the diet increased the proportion of 18:2n-6 in Daphnia, but the proportion of 20:4n-6 was not affected. Daphnia supplemented with 22:5n-6 contained ~3.5% 20:4n-6 in the lipids and FA specific stable isotope analyses validated that the increase in the proportion of 20:4n-6 was due to retroconversion of unlabeled 22:5n-6. These results suggest that chain shortening of 22:5n-6 is a more efficient pathway to synthesize 20:4n-6 in D. magna than elongation and desaturation of 18:2n-6. These results may at least partially explain the discrepancies noticed between phytoplankton FA composition and the expected FA composition in freshwater cladocerans. Finally, retroconversion of dietary 22:5n-6 to 20:4n-6 indicates Daphnia efficiently retain long chain n-6 FA in lake food webs, which might be important for the nutritional ecology of fish.

Metabolic products of soluble epoxide hydrolase are essential for monocyte chemotaxis to MCP-1 in vitro and in vivo.

Monocyte chemoattractant protein-1 (MCP-1)-induced monocyte chemotaxis is a major event in inflammatory disease. Our prior studies have demonstrated that MCP-1-dependent chemotaxis requires release of arachidonic acid (AA) by activated cytosolic phospholipase A(2) (cPLA(2)). Here we investigated the involvement of AA metabolites in chemotaxis. Neither cyclooxygenase nor lipoxygenase pathways were required, whereas pharmacologic inhibitors of both the cytochrome-P450 (CYP) and the soluble epoxide hydrolase (sEH) pathways blocked monocyte chemotaxis to MCP-1. To verify specificity, we demonstrated that the CYP and sEH products epoxyeiscosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs), respectively, restored chemotaxis in the presence of the inhibitors, indicating that sEH-derived products are essential for MCP-1-driven chemotaxis. Importantly, DHETs also rescued chemotaxis in cPLA(2)-deficient monocytes and monocytes with blocked Erk1/2 activity, because Erk controls cPLA(2) activation. The in vitro findings regarding the involvement of CYP/sEH pathways were further validated in vivo using two complementary approaches measuring MCP-1-dependent chemotaxis in mice. These observations reveal the importance of sEH in MCP-1-regulated monocyte chemotaxis and may explain the observed therapeutic value of sEH inhibitors in treatment of inflammatory diseases, cardiovascular diseases, pain, and even carcinogenesis. Their effectiveness, often attributed to increasing EET levels, is probably influenced by the impairment of DHET formation and inhibition of chemotaxis.

Pythium irregulare fermentation to produce arachidonic acid (ARA) and eicosapentaenoic acid (EPA) using soybean processing co-products as substrates.

Arachidonic acid (ARA) and eicosapentaenoic acid (EPA) were produced by Pythium irregulare fungus using soybean cotyledon fiber and soy skim, two co-products from soybean aqueous processing, as substrates in different fermentation systems. Parameters such as moisture content, substrate glucose addition, incubation time, and vegetable oil supplementation were found to be important in solid-state fermentation (SSF) of soybean fiber, which is to be used as animal feed with enriched long-chain polyunsaturated fatty acids (PUFA). Soybean fiber with 8 % (dwb) glucose supplementation for a 7-day SSF produced 1.3 mg of ARA and 1.6 mg of EPA in 1 g of dried substrate. When soy skim was used as substrate for submerged fermentation, total ARA yield of 125.7 mg/L and EPA yield of 92.4 mg/L were achieved with the supplementation of 7 % (w/v) soybean oil. This study demonstrates that the values of soybean fiber and soy skim co-products could be enhanced through the long-chain PUFA production by fermentation.

Linoleic acid supplementation results in increased arachidonic acid and eicosanoid production in CF airway cells and in cftr-/- transgenic mice.

Cystic fibrosis (CF) patients display a fatty acid imbalance characterized by low linoleic acid levels and variable changes in arachidonic acid. This led to the recommendation that CF patients consume a high-fat diet containing >6% linoleic acid. We hypothesized that increased conversion of linoleic acid to arachidonic acid in CF leads to increased levels of arachidonate-derived proinflammatory metabolites and that this process is exacerbated by increasing linoleic acid levels in the diet. To test this hypothesis, we determined the effect of linoleic acid supplementation on downstream proinflammatory biomarkers in two CF models: 1) in vitro cell culture model using 16HBE14o(-) sense [wild-type (WT)] and antisense (CF) human airway epithelial cells; and 2) in an in vivo model using cftr(-/-) transgenic mice. Fatty acids were analyzed by gas chromatography-mass spectrometry (GC/MS), and IL-8 and eicosanoids were measured by ELISA. Neutrophils were quantified in bronchoalveolar lavage fluid from knockout mice following linoleic acid supplementation and exposure to aerosolized Pseudomonas LPS. Linoleic acid supplementation increased arachidonic acid levels in CF but not WT cells. IL-8, PGE(2), and PGF(2α) secretion were increased in CF compared with WT cells, with a further increase following linoleic acid supplementation. cftr(-/-) Mice supplemented with 100 mg of linoleic acid had increased arachidonic acid levels in lung tissue associated with increased neutrophil infiltration into the airway compared with control mice. These findings support the hypothesis that increasing linoleic acid levels in the setting of loss of cystic fibrosis transmembrane conductance regulator (CFTR) function leads to increased arachidonic acid levels and proinflammatory mediators.

Effect of dietary levels of corn oil on maternal arachidonic acid synthesis and fatty acid composition in lactating rats.

OBJECTIVE: We examined the effect of different amounts of dietary corn oil rich in linoleic acid (LA) on the endogenous synthesis of arachidonic acid (AA), uptake of its precursor LA, and fatty acid composition of tissues involved in the supply of long-chain polyunsaturated fatty acids for milk synthesis. METHODS: Female Sprague Dawley rats received one of the following diets during pregnancy and lactation: a low-lipid diet (LLD; 2%), an adequate-lipid diet (ALD; 5%), or a high-lipid diet (HLD; 10%). Lipids were provided by corn oil. On day 12 of lactation we measured the endogenous synthesis of AA and quantified the conversion of (13)C-LA to (13)C-AA and the metabolic fate of (13)C-LA from all dietary groups. RESULTS: The LLD rats demonstrated larger amounts of endogenous synthesis of (13)C-AA and more dietary (13)C-LA transferred to the mammary gland (MG) than HLD rats during lactation. The proportion of medium-chain fatty acids was higher in the MG, milk clot, and liver of LLD than of HLD rats. Daily volume and 24-h yield of lipids and energy were lower in LLD rats than in HLD rats. Measurements of milk composition demonstrated that fat concentration significantly increased as lipid concentration increased in the diet. CONCLUSION: These results suggest that maternal adaptations used to compensate for diets deficient in long-chain polyunsaturated fatty acids include increased endogenous synthesis of AA and elevated uptake of LA in the MG and increased synthesis of medium-chain polyunsaturated fatty acids. It appears that the MG and liver participate together for AA synthesis for milk when this fatty acid is not provided in the diet.

Fatty acid composition abnormalities in atopic disease: evidence explored and role in the disease process examined.

Summary There is a hypothesis causally linking excess intake of n-6 polyunsaturated fatty acids (PUFAs) to atopic disease. Under most dietary conditions, the main precursor of eicosanoids is the n-6 PUFA arachidonic acid (AA). AA-derived eicosanoids play many roles in sensitization to allergens and in allergic inflammation. Long chain n-3 PUFAs inhibit AA incorporation into cell membranes and inhibit AA metabolism to eicosanoids. It is hypothesized that atopy is associated with a higher n-6 PUFA status and with a low n-3 PUFA status. However, measurements of fatty acid composition do not provide a clear picture that such fatty acid abnormalities exist in atopy with no really clear pattern of altered status of a particular fatty acid or a particular fatty acid family. There are few reports of elevated linoleic acid in atopy. Some studies report lower amounts of the n-6 PUFAs, including AA, and of long chain n-3 PUFAs in atopy, although observations on this are not consistent. Taken together these data clearly do not support the hypothesis that atopy is somehow associated with a high exposure to, and status of, n-6 PUFAs. Intervention studies with n-3 PUFAs in pregnant women, infants and children suggest some clinical benefits, although how long lasting these are remains to be determined. The observation that there may be low AA status in atopy suggests that fish oil intervention, which targets AA status and metabolism, may not be ideal and that a combination of fish oil with some longer chain n-6 PUFAs may be more efficacious.

Synthesis of long-chain polyunsaturated fatty acids in preterm newborns fed formula with long-chain polyunsaturated fatty acids.

BACKGROUND: Docosahexaenoic acid (DHA) and arachidonic acid (AA) are long-chain polyunsaturated fatty acids (LCPs) that play pivotal roles in growth and neurodevelopment. OBJECTIVE: We aimed to quantify the synthesis of LCPs in preterm infants fed infant formula containing LCPs. DESIGN: Twenty-two preterm infants were randomly assigned to either the no-LCP group (fed formula without LCPs; n = 11) or the LCP group (fed formula with LCPs; n = 11). Dietary LCPs had higher (13)C content than did the endogenously synthesized LCPs, which were derived from linoleic and alpha-linolenic acids. The (13)C content of major selected plasma fatty acids was measured by using gas chromatography-isotope ratio mass spectrometry at birth and at age 1, 3, and 7 mo. Absolute LCP synthesis and the percentage of LCP synthesis relative to dietary intake were calculated. RESULTS: Percentage AA synthesis was 67.2 +/- 7.8%, 35.9 +/- 9.8%, and 29.0 +/- 10.3%, and that of DHA was 41.7 +/- 14.9%, 10.5 +/- 8.1%, and 7.4 +/- 6.2% at 1, 3, and 7 mo old, respectively. Absolute AA synthesis was 26.7 +/- 4.2, 14.4 +/- 3.9, and 11.6 +/- 4.1 mg x kg(-1) x d(-1) and that of DHA was 12.6 +/- 4.5, 3.2 +/- 2.5, and 2.3 +/- 1.9 mg x kg(-1) . d(-1) at 1, 3, and 7 mo old, respectively. AA and DHA synthesis decreased significantly (P < 0.01) with time, and AA synthesis was significantly (P < 0.01) greater than DHA synthesis. CONCLUSIONS: By this novel approach, we measured endogenous LCP synthesis in infants receiving dietary LCPs over long periods. By age 7 mo, LCP synthesis was dramatically lower in preterm infants fed LCPs.

Identification and functional characterization of the moss Physcomitrella patens delta5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis.

The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. Here we report the identification and characterization of a delta5-desaturase from P. patens that is associated with the synthesis of these fatty acids. A full-length cDNA for this desaturase was identified by data base searches based on homology to sequences of known delta5-desaturase cDNAs from fungal and algal species. The resulting P. patens cDNA encodes a 480-amino acid polypeptide that contains a predicted N-terminal cytochrome b5-like domain as well as three histidine-rich domains. Expression of the enzyme in Saccharomyces cerevisiae resulted in the production of the delta5-containing fatty acid arachidonic acid in cells that were provided di-homo-gamma-linolenic acid. In addition, the expressed enzyme generated delta5-desaturation products with the C20 substrates omega-6 eicosadienoic and omega-3 eicosatrienoic acids, but no products were detected with the C18 fatty acid linoleic and alpha-linolenic acids or with the C22 fatty acid adrenic and docosapentaenoic acids. When the corresponding P. patens genomic sequence was disrupted by replacement through homologous recombination, a dramatic alteration in the fatty acid composition was observed, i.e. an increase in di-homo-gamma-linolenic and eicosatetraenoic acids accompanied by a concomitant disappearance of the delta5-fatty acid arachidonic and eicosapentaenoic acids. In addition, overexpression of the P. patens cDNA in protoplasts isolated from a disrupted line resulted in the restoration of arachidonic acid synthesis.

[Isolation and functional identification of delta 5 desaturase gene from Mortierella alpina].

Arachidonic acid is an essential fatty acid in human nutrition and a biogenetic precursor of the biologically active prostaglandins and leukotrienes. delta5 desaturase is a key enzyme in the biosynthetic pathway of arachidonic acid, which catalyze the delta5 dehydrogenation of di-homo-gamma-linolenic acid to form arachidonic acid. Complete cDNA encoding putative delta5 fatty acyl desaturase was isolated from Mortierella alpina M6 via RT-PCR. The full length cDNA consisted of 1366 nucleotides encoding 446 amino acids. The deduced protein had conserved domains of known delta5 fatty acyl desaturases including a cytochrome b5 domain in the N terminal and 3 conserved histidine box. To elucidate the function of the protein, the cDNA was subcloned into the expression vector pPIC9K. The resultant recombinant plasmid pPIC9K-D5 was transformed to Pichia pastoris GS115 by electroporation. Transformants containing multi-copy delta5 desaturase gene were screened by Geneticin resistance. The transformants were induced to express the inserted gene with methanol when di-homo-gamma-linolenic acid was provided as an exogenous substrate. Analysis of the recombinant yeast lipids by gas chromatograph showed that a novel peak corresponding to the standard of arachidonic acid was detected. The novel peak was further characterized by GC-MS and identified as arachidonic acid. The results showed that the gene isolated from fungi Mortierella alpina M6 was a delta5 desaturase gene.

Increased arachidonic acid concentration in the brain of Flinders Sensitive Line rats, an animal model of depression.

Depression may be associated with impaired membrane PUFA composition, especially decreased n-3 PUFA. This assumption has not been tested at the level of brain tissue. Moreover, most studies were confounded by dietary variability. We examined the FA composition of selected brain areas in an animal model of depression, the Flinders Sensitive Line (FSL) rat, and compared the findings with those in controls fed identical diets. In all brain regions studied, the concentration of arachidonic acid (AA) was significantly higher in the FSL rats: in the hypothalamus by 21%, in the nucleus accumbens by 24%, in the prefrontal cortex by 31%, and in the striatum by 23%. No significant differences were observed for n-3 PUFA or for the saturated and monounsaturated FAs. Our results confirm the existence of altered brain PUFA composition in an animal model of depression. The finding of increased AA, an n-6 PUFA, rather than decreased n-3 PUFA, emphasizes the importance of both PUFA families in the pathophysiological processes underlying depression. The FSL rat is a useful tool for further elucidation of the FA disturbances in depression.