Oxidative pathways of arachidonic acid as targets for regulation of platelet activation.

Platelet activation plays an important role in acute and chronic cardiovascular disease states. Multiple pathways contribute to platelet activation including those dependent upon arachidonic acid. Arachidonic acid is released from the platelet membrane by phospholipase A2 action and is then metabolized in the cytosol by specific arachidonic acid oxidation enzymes including prostaglandin H synthase, 12-lipoxygenase, and cytochrome P450 to produce pro- and anti-inflammatory eicosanoids. This review aims to analyze the role of arachidonic acid oxidation on platelet activation, the enzymes that use it as a substrate associated as novel therapeutics target for antiplatelet drugs.

Changes in the Lipid Composition of Biological Membranes under the Influence of Endogenous and Exogenous Factors.

Quantitative and qualitative assessments of cell membrane components are essential for the accurate interpretation of processes occurring in biological membranes. Changes in the structure and function of cell membrane components have been linked to oxidative stress. Oxidative stress induced by chronic ethanol consumption or cancer transformation has been implicated in changing the levels of phospholipids and fatty acids in the cell membrane. In this study, we used high-performance liquid chromatography to quantitate the effects of alcohol and malignant transformation on membrane components, namely phospholipids and free fatty acids. Ethanol increased the phospholipid levels. Moreover, the process of malignant transformation was accompanied by increased levels of phospholipids and arachidonic acid as well as decreased levels of linoleic acid and α-linolenic acid. Thus, these oxidative stress-inducing conditions that cause variations in the cellular composition affect the actions of the cell membrane and cell function.

An improved Ultra-High Performance Liquid chromatography-tandem mass spectrometry method for simultaneous quantitation of cytochrome P450 metabolites of arachidonic acid in human plasma.

This study aims to develop a straightforward, sensitive UHPLC-MS/MS method to quantify 15 eicosanoids derived from arachidonic acid in human plasma. Tert-Butyl methyl ether was used on the liquid-liquid extraction method and significantly reduced the expense and time. The method showed excellent linearity for all analytes, with regression coefficients higher than 0.99 over a wide range of concentrations from 0.01 ng mL-1 to 100 ng mL-1. The recovery rates were over 65.00%, and the matrix effects ranged from 8.42% to 40.00%. The limits of detection ranged from 6 pg mL-1 to 10 pg mL-1, and all of the limits of quantification were 20 - 33 pg mL-1. For the broad concentration range, the RE% for accuracy and precision were less than ±â€¯15%. Moreover, trans-4-{4-[3-(4-Trifluoromethoxyphenyl)-ureido] cyclohexyloxy} benzoic acid (t-TUCB) pretreatment extended the window of detection for as much as 30 days. Eicosanoid signaling is altered in various neurological diseases, including pain, Alzheimer's disease and major depressive disorder. Therefore, this rapid, robust quantitative profiling of 15 eicosanoids in plasma could provide a distinct eicosanoid fingerprint for precision medicine in these patients.

Single-tube biosynthesis and extraction of U-13C and U-14C arachidonic acid from microcultures of Mortierella alpina for in vivo pharmacology and metabolic tracing studies.

Heavy and radioisotope labeling are commonly used methods to trace the pharmacological activity and metabolic fate of a biochemical or pharmaceutical in vivo. Recent years witnessed increased demand for molecules uniformly labeled with heavy carbon-13 (U-13C) or radioactive carbon-14 (U-14C) isotopes over singly labeled isotopic versions. Polyunsaturated fatty acids (PUFAs) represent one classic example where uniform 13C or 14C isotopic enrichment of the hydrocarbon backbone has numerous technical, metabolic and pharmacological advantages. PUFAs are usually produced in fungi or algae and uniform 13C or 14C enrichment of the hydrocarbon chain is achieved by feeding the microorganism a suitable U-13C or U-14C substrate. Previous literature methods describing the biosynthesis of U-13C or U-14C fatty acids reported variable isotopic enrichments that were less than anticipated and suffered from inconsistent growth of the microorganism due to radiotoxicity. In the present study, a single-tube method is described for the biosynthesis and extraction of U-13C and U-14C arachidonic acid (AA), a standard PUFA, from microcultures of the soil fungus Mortierella alpina. To produce U-13C-AA, a suspension of fungal spores and mycelial fragments was directly inoculated and grown into submerged cultures in a medium composed of U-13C-glucose and NaNO3 as the respective and only sources of carbon and nitrogen. The total 13C enrichment of AA was in excess of 95% and the percentage of U-13C-AA was in the range of 60-70%. These values have not been surpassed by previously reported methods. To produce U-14C-AA, the procedure was modified to limit the radiotoxic effects of 14C on fungal growth. Submerged cultures were initially grown on common 12C-glucose. Then, following glucose depletion, the biomass was collected and immediately cultured on U-14C-glucose. This approach is unprecedented in reported literature and has significantly limited the radiotoxic effects of 14C on the microorganism. Biomass transfer from 12C to 14C substrates was timed to keep an uninterrupted supply of carbon required to sustain the microorganism in the fatty acid synthesis mode and suppress ß-oxidation, a metabolic status that is prerequisite for enhanced isotopic purity of the 14C product. The specific activity of 14C enriched AA was estimated at 864 Ci/mol (range of 708-1020 Ci/mol) suggesting 69.2% (range of 56.7-81.7%) 14C enrichment along the AA hydrocarbon backbone. The present method used a single tube for microbial culture and lipid extraction to minimize manipulative losses and oxidative degradation of the labeled products. Production cost is comparatively cheaper to custom labeling and yields of U-13C and U-14C-AA are comparable to literature methods and sufficient for small scale in vitro and in vivo pharmacological studies.

Long chain fatty acids and related pro-inflammatory, specialized pro-resolving lipid mediators and their intermediates in preterm human milk during the first month of lactation.

This study aimed to measure longitudinal quantities of the long chain fatty acids, their biologically active terminal metabolites and related intermediates (also called oxylipins) in preterm human milk expressed during the first month of lactation. In a prospective cohort, breast milk was collected throughout the first month of lactation in 30 women who delivered preterm infants. Eighteen bioactive lipids and their intermediates were quantified via solid phase extraction and LC-MS/MS. Analysis by GC-FID quantified the fatty acid precursors. Arachidonic acid (ARA) and docosahexaenoic acid (DHA) milk concentrations significantly declined throughout the first month. Oxylipin concentrations did not change during lactation. Positive associations existed between ARA and thromboxane B2, eicosapentaenoic acid and 18-hydroxyeicosapentaenoic acid, and between DHA and PDX and 14- and 17-hydroxydocosahexaenoic acids. DHA concentrations were 1.5 times higher and 14-HDHA was 1.7 times higher in milk from women taking DHA supplements. This investigation showed conditionally essential fatty acids, ARA and DHA, decreased in preterm milk, suggesting a need to supplement their intake for the breast milk-fed preterm infant. Positive associations between parent fatty acids, bioactive lipids and intermediates, as well as sensitivity of milk to maternal fatty acid intake, support consideration of a comprehensive approach to providing fatty acids for preterm infants through both maternal and infant supplementation.

The Distribution of Phosphatidylcholine Species in Superficial-Type Pharyngeal Carcinoma.

Objectives. Superficial-type pharyngeal squamous cell carcinoma (STPSCC) is defined as carcinoma in situ or microinvasive squamous cell carcinoma without invasion to the muscular layer. An exploration of the biological characteristics of STPSCC could uncover the invasion mechanism of this carcinoma. Phosphatidylcholine (PC) in combination with fatty acids is considered to play an important role in cell motility. Imaging mass spectrometry (IMS) is especially suitable for phospholipid analysis because this technique can distinguish even fatty acid compositions. Study Design. IMS analysis of frozen human specimens. Methods. IMS analysis was conducted to elucidate the distribution of PC species in STPSCC tissues. STPSCC tissue sections from five patients were analyzed, and we identified the signals that showed significant increases in the subepithelial invasive region relative to the superficial region. Results. Three kinds of PC species containing arachidonic acid, that is, PC (16:0/20:4), PC (18:1/20:4), and PC (18:0/20:4), were increased in the subepithelial invasive region. Conclusion. These results may be associated with the invasion mechanism of hypopharyngeal carcinoma.

Secreted Phospholipase A2 Specificity on Natural Membrane Phospholipids.

The secreted phospholipase A2 (sPLA2) family contains 10 catalytically active isoforms. Current in vitro biochemical studies have shown that individual sPLA2s have distinct substrate selectivity in terms of the polar head groups or sn-2 fatty acids of their substrate phospholipids. Importantly, transgenic or knockout mice for distinct sPLA2s display nonoverlapping phenotypes, arguing that they do act on different phospholipid substrates and mobilize unique lipid metabolites in vivo. In an effort to comprehensively understand lipid metabolism driven by individual sPLA2s under pathophysiological conditions, we took advantages of mass spectrometric lipidomics technology to monitor the spatiotemporal changes in phospholipids (substrates) and products (fatty acids, lysophospholipids, and their metabolites) in tissues or cells of sPLA2-transgenic or knockout mice. The in vivo lipidomic data were compared with the in vitro activity of recombinant sPLA2s toward phospholipid mixtures extracted from the target tissues, cells, or extracellular membrane components on which sPLA2s may intrinsically act. These approaches reveal that the overall tendency in in vitro assays using natural membranes is recapitulated in several in vivo systems, often with even more selective patterns of hydrolysis. In this chapter, we will summarize current understanding of the in vivo substrate specificity of sPLA2s toward natural membrane phospholipids.

Targeted profiling of arachidonic acid and eicosanoids in rat tissue by UFLC-MS/MS: Application to identify potential markers for rheumatoid arthritis.

We describe a method for the targeted analysis of bioactive arachidonic acid metabolites through cyclooxygenase (COX) and lipoxygenase (LOX) pathway in knee joint, liver, kidney, spleen and heart using an ultra-fast liquid chromatography-tandem mass (UFLC-MS/MS) method. Method validation was investigated, including linearity, precision, accuracy, matrix effect, extraction recovery and stability for the simultaneous analysis of prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). The method enables us to chromatographically separate branched-chain species from their straight-chain isomers as well as separate biologically important eicosanoids. The concentrations of the following major eicosanoids were significantly increased in rheumatoid arthritis model rats than in normal ones: 5-HETE, 8-HETE, 12-HETE, 15-HETE, PGF2α, TXB2, 5-HpETE, LTE4, PGE2, PGD2, LTB4. Further multivariate data analysis (partial least square-discriminant analysis) showed COX products (PGs, TXs) were readily distributed towards liver and kidney, LOX products (LTs, HETEs) towards knee joint and spleen, and heart had no characteristic metabolites. The method described here offers a useful tool for the evaluation of complex regulatory eicosanoids responses in RA disease states and provides support for use of dual inhibitors of COX and LOX enzymes on RA treatment.

The Association of Fatty Acid Levels and Gleason Grade among Men Undergoing Radical Prostatectomy.

BACKGROUND: Epidemiological data suggest that omega-6 (ω-6) fatty acids (FAs) may be associated with cancer incidence and/or cancer mortality, whereas ω-3 FAs are potentially protective. We examined the association of the ratio of ω-6 to ω-3 FA (ω-6:ω-3) and individual FA components with pathological results among men with prostate cancer (PCa) undergoing radical prostatectomy. METHODS: Sixty-nine men were included in the study. Components of ω-6 (linoleic acid (LA), arachidonic acid (AA), and dihomo-γ-linolenic acid (DGLA)) and ω-3 (docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)) were analyzed by liquid chromatography/mass selective detector separation. Logistic regression analysis was performed to determine association of FA with pathological high grade (Gleason ≥4+3) disease. RESULTS: The were 35 men with low grade disease (Gleason ≤3+4) and 34 men with high grade disease. Men with low grade disease were significantly younger (58y vs 61y, p = 0.012) and had lower D'Amico clinical classification (p = 0.001) compared to men with high grade disease. There was no significant association of ω-6:ω-3 with high grade disease (OR 0.93, p = 0.78), however overall ω-6, ω-3, and individual components of ω-6 and ω-3 FAs except EPA were significantly associated with high grade disease (ω-6: OR 3.37, 95% CI: 1.27,8.98; LA: OR 3.33, 95% CI:1.24,8.94; AA: OR 2.93, 95% CI:1.24,6.94; DGLA: OR 3.21, 95% CI:1.28,8.04; ω-3: OR 3.47, 95% CI:1.22,9.83; DHA: OR 3.13, 95% CI:1.26,7.74). ω-6 and ω-3 FA components were highly correlated (Spearman ρ = 0.77). CONCLUSION: Higher levels of individual components of ω-6 and ω-3FAs may be associated with higher-grade PCa. IMPACT: Studies into the causative factors/pathways regarding FAs and prostate carcinogenesis may prove a potential association with PCa aggressiveness.

Drosophila Fed ARA and EPA Yields Eicosanoids, 15S-Hydroxy-5Z,8Z, 11Z, 13E-Eicosatetraenoic Acid, and 15S-Hydroxy-5Z,8Z,11Z,13E,17Z-Eicosapentaenoic Acid.

Drosophila melanogaster has been a widely used as a model system for its powerful genetic tools. However, it remains to be illustrated if Drosophila can be used to examine the biochemical and physiological metabolism of eicosanoids. Thus, the analysis on the metabolism of C20 polyunsaturated fatty acids (PUFA) in Drosophila was implemented with high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Fatty acid (FA) analysis of the whole body, head, and thorax-abdomen in Drosophila showed C20 PUFA could only be found in Drosophila fed diets supplemented with eicosapentaenoic acid (EPA) and arachidonic acid (ARA), but not in Drosophila fed base diets. The C20 PUFA were found in abundance in the head. Drosophila fed ARA- and EPA-supplemented diets yielded 15S-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid [15(S)-HETE] and 15S-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid [15(S)-HEPE], respectively, while other sampled eicosanoids could not be detected. Similar results were obtained by incubating fly tissue supplemented with ARA or EPA. Furthermore, a genome sequence scan indicated that no gene encoding the key enzymes synthesizing eicosanoids were found in Drosophila. These findings demonstrate that Drosophila may possess a special lipid metabolic system, which is different from mammals.

CYTOTOXIC EFFECTS OF THE RED SEA SOFT CORAL SARCOPHYTON TROCHELIOPHORUM.

The present study describes the in vitro cytotoxic effects of soft coral (Sarcophyton tiocheliophorum). Soft corals of genus Sarcophyton were reported to contain compounds that are active against brine shrimp and promote paclitaxel cytotoxicity in the human colon cancer Caco-2 cell line. The n-hexane extract of the soft coral Sarcophyton tiocheliophorum induced significant dose-dependent toxicity (LC50 96.7 ppm) compared with ethyl acetate (LC50. 120 ppm). We reported the most active cytotoxic level to be correspondence to LC50 values of 20.2, 59.2 ppm and 18.9 and 26 ppm. Accordingly, bio-assay guided fractionation was conducted to identi- fy the bioactive compounds. Arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid were characterized based on GC-MS analyses. Our results demonstrate the value of marine products as a natural source of medicinally interesting cytotoxic compounds.

Breast milk from women living near Lake Malawi is high in docosahexaenoic acid and arachidonic acid.

Adequate long-chain polyunsaturated fatty acid (LCPUFA) intake is critical during the fetal and infant periods. We quantified fatty acid content of breast milk (n=718) and plasma from six month old infants (n=412) in southern Malawi, and in usipa (n=3), a small dried fish from Lake Malawi. Compared to global norms, Malawian breast milk fatty acid content (% of total fatty acids) was well above average levels of arachidonic acid [ARA] (0.69% vs. 0.47%) and docosahexaenoic acid [DHA] (0.73% vs. 0.32%). Average Malawian infant plasma ARA (7.5%) and DHA (3.8%) levels were comparable to those reported in infants consuming breast milk with similar fatty acid content. The amounts (mg) of DHA, EPA and ARA provided by a 3 oz (85 g) portion of dried usipa (1439, 659 and 360, respectively) are considerably higher than those for dried salmon. Usipa may be an important source of LCPUFA for populations in this region.

Novel liquid chromatography-mass spectrometry method shows that vitamin E deficiency depletes arachidonic and docosahexaenoic acids in zebrafish (Danio rerio) embryos.

To test the hypothesis that embryogenesis depends upon α-tocopherol (E) to protect embryo polyunsaturated fatty acids (PUFAs) from lipid peroxidation, new methodologies were applied to measure α-tocopherol and fatty acids in extracts from saponified zebrafish embryos. A solid phase extraction method was developed to separate the analyte classes, using a mixed mode cartridge (reverse phase, π-π bonding, strong anion exchange), then α-tocopherol and cholesterol were measured using standard techniques, while the fatty acids were quantitated using a novel, reverse phase liquid chromatography-mass spectrometry (LC-MS) approach. We also determined if α-tocopherol status alters embryonic lipid peroxidation products by analyzing 24 different oxidized products of arachidonic or docosahexaenoic (DHA) acids in embryos using LC with hybrid quadrupole-time of flight MS. Adult zebrafish were fed E- or E+ diets for 4 months, and then were spawned to obtain E- and E+ embryos. Between 24 and 72 hours post-fertilization (hpf), arachidonic acid decreased 3-times faster in E- (21 pg/h) compared with E+ embryos (7 pg/h, P<0.0001), while both α-tocopherol and DHA concentrations decreased only in E- embryos. At 36 hpf, E- embryos contained double the 5-hydroxy-eicosatetraenoic acids and 7-hydroxy-DHA concentrations, while other hydroxy-lipids remained unchanged. Vitamin E deficiency during embryogenesis depleted DHA and arachidonic acid, and increased hydroxy-fatty acids derived from these PUFA, suggesting that α-tocopherol is necessary to protect these critical fatty acids.

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.

[Biosynthesis of arachidonic acid by micromycetes (review)].

Arachidonic acid (ARA, 5,8,11,14-cis-eicosatetraenoic acid) is widely used in medicine, pharmaceutics, cosmetics, dietary nutrition, agriculture, and other fields. Microbiological production of ARA is of increased interest since the natural sources (pig liver, adrenal glands, and egg-yolk) cannot satisfy its growing requirements. Mechanisms for ARA biosynthesis as well as the regulation of enzymes involved in this process are considered. Review summarizes literature data concerning individual stages of microbiological ARA production, methods for screening of active strains-producers, physiological regulation of ARA synthesis in micromycetes (the effect of growth phase, medium composition, pH, temperature, and aeration), and effective technologies of fermentation and the product recovery. Information on the whole biotechnological process from strain selection to the ARA yield improvement and purification of the end product is presented.

Safety evaluation of arachidonic acid rich Mortierella alpina biomass in albino rats--a subchronic study.

Safety evaluation of arachidonic acid rich Mortierella alpina biomass was carried out in Wistar rats by acute and subchronic oral toxicity studies. A preliminary acute toxicity study revealed that the biomass was safe at acute doses and that the LD50 exceeded 5000mg/kg BW, the highest dose used in the study. In subchronic study, rats were fed diet containing 0, 2500, 5000, 10,000, 20,000 and 30,000mg/kg, M. alpina biomass for a period of 13 weeks. Results indicated that biomass fortification had a positive influence on growth with no overt toxic effects on the survival, food consumption and body weight gain throughout the treatment interlude. The statistically significant changes in relative organ weights, serum biochemical and hematological indices in M. alpina fed groups' viz., higher relative weights of spleen, liver, brain and ovary in females, reduced hemoglobin concentration in males, elevated WBC counts at highest dose, reduction in serum triglycerides and increased alkaline phosphatase activity were not concomitant with pertinent histopathological changes and hence toxicologically inconsequential. No microscopic or macroscopic lesions attributable to the treatment were manifested in the experimental groups. The results of the present study strongly advocate the safety of M. alpina biomass in rats at levels used in the study.

[Primary role and extraction of arachidonic acid].

Arachidonic acid (AA), which is one of the essential fatty acids in the human body, plays an important physiological, pharmacological and health role. This paper discusses the general characteristics of arachidonic acid, physiological, pharmacological effects and health roles. There are also a comparison of arachidonic acid extraction methods and domestic developments and problems existed. At last, it is prospected the trend of its development.

Separation and preparative purification of arachidonic acid from microbial lipids by urea inclusion reaction and HPLC.

Arachidonic acid (AA) was separated and purified from microbial lipids by the combined method of urea inclusion reaction and reversed-phase high performance liquid chromatography. At first, AA was concentrated from free fatty acids made from microbial lipids by a urea inclusion reaction. The optimum conditions were as follows: methanol was the suitable solvent, the ratio of free fatty acids to urea to methanol was 1:2:8 (wt/wt), and the temperature of the urea inclusion reaction was -10 degrees C. The AA content was increased from 38% to 79%, and then AA was purified on a C(18) preparative column (300 mm x 30 mm I.D., d(p)=15 microm), using methanol-water (95:5, v/v) as the mobile phase, at a flow rate of 5 mL/min. The purity of AA after two steps purification reached 99%. This result indicates that the combined method of the urea inclusion reaction and reversed-phase high performance liquid chromatography is a promising technique for purification of AA.

Cytochrome P450/NADPH-dependent formation of trans epoxides from trans-arachidonic acids.

Trans-arachidonic acids (trans-AA) are products of cis-trans isomerization of arachidonic acid by nitrogen dioxide radical (NO(2)), and occur in vivo, but their metabolism is unknown. We found that hepatic microsomes oxidized trans-AA via cytochrome P450/NADPH system to epoxides, which were hydrolyzed by epoxide hydrolase to diols (DiHETEs). 14,15-trans-AA produced one erythro diol and three threo diols each having one trans double bond.

Diatom cultivation and biotechnologically relevant products. Part II: current and putative products.

While diatoms are widely present in terms of diversity and abundance in nature, few species are currently used for biotechnologically applications. Most studies have focussed on intracellularly synthesised eicosapentaenoic acid (EPA), a polyunsaturated fatty acid (PUFA) used for pharmaceutical applications. Applications for other intracellular molecules, such as total lipids for biodiesel, amino acids for cosmetic, antibiotics and antiproliferative agents, are at the early stage of development. In addition, the active principle component must be identified amongst the many compounds of biotechnological interest. Biomass from diatom culture may be applied to: (1). aquaculture diets, due to the lipid- and amino-acid-rich cell contents of these microorganisms, and (2). the treatment of water contaminated by phosphorus and nitrogen in aquaculture effluent, or heavy metal (bioremediation). The most original application of microalgal biomass, and specifically diatoms, is the use of silicon derived from frustules in nanotechnology. The competitiveness of biotechnologically relevant products from diatoms will depend on their cost of production. Apart from EPA, which is less expensive when obtained from Phaeodactylum tricornutum than from cod liver, comparative economic studies of other diatom-derived products as well as optimisation of culture conditions are needed. Extraction of intracellular metabolites should be also optimised to reduce production costs, as has already been shown for EPA. Using cell immobilisation techniques, benthic diatoms can be cultivated more efficiently allowing new, biotechnologically relevant products to be investigated.