Satouchi, K. Lysophosphatidylcholine from white muscle of bonito Euthynnus pelamis Linnaeus : Involvement of phospholipase A1 activity for its production. Acta , — Saulnier-Blache, J. A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification. Lipid Res. Shen, Z. Fatty acid composition of lysophosphatidic acid and lysophosphatidylinositol in plasma from patients with ovarian cancer and other gynecological diseases. Sliva, D. Enhancement of the migration of metastatic human breast cancer cells by phosphatidic acid.
Spiegel, S. Sphingosinephosphate: An enigmatic signalling lipid. Stracke, M. Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. Sugo, T. Identification of a lysophosphatidylserine receptor on mast cells. Sutphen, R. Lysophospholipids are potential biomarkers of ovarian cancer. Cancer Epidemiol.
Biomarkers Prev. Tanaka, T. Quantitative analysis of lysophosphatidic acid by time-of-flight mass spectrometry using a phosphate-capture molecule. Tanyi, J. Role of decreased levels of lipid phosphate phosphatase-1 in accumulation of lysophosphatidic acid in ovarian cancer. The human lipid phosphate phosphatase-3 decreases the growth, survival, and tumorigenesis of ovarian cancer cells: Validation of the lysophosphatidic acid signaling cascade as a target for therapy in ovarian cancer.
Tokumura, A. Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase. Lack of significant differences in the corrected activity of lysophospholipase D, producer of phospholipid mediator lysophosphatidic acid, in incubated serum from women with and without ovarian tumors.
Cancer 94, — Umezu-Goto, M. Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production. Lysophosphatidic acid productio anaction: Validated targets in cancer? Cell Biochem. Van Brocklyn, J. Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: Roles of sphingosine kinase isoforms in growth of glioblastoma cell lines.
Inhibition of autotaxin by lysophosphatidic acid and sphingosine 1-phosphate. Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development. Visentin, B. Cacer Cell 9, — Xiao, Y. Evaluation of plasma lysophospholipids for diagnostic significance using electrospray ionization mass spectrometry ESI-MS analyses. NY Acad. Electrospray ionization mass spectrometry analysis of lysophospholipids in human ascitic fluids: Comparison of the lysophospholipid contents in malignant vs nonmalignant ascitic fluids. Xu, Y. Lysophospholipids activate ovarian and breast cancer cells.
Lysophosphatidic acid as a potential biomarker for ovarian and other gynecologic cancers. Expression of autotaxin NPP-2 is closely linked to invasiveness of breast cancer cells. Metastasis 19, — Yang, Y. Autotaxin expression in non—small-cell lung cancer. Cell Mol. Zhang, G. Expression of autotaxin mRNA in human hepatocellular carcinoma. Introduction 1. COX pathway 1. LOX pathway 1. Cytochrome P pathway 2.
Quantitative Measurement of Eicosanoids 3. Preparation of Samples 3. Urine samples 3. Plasma preparation 3. Collection of intestinal tumor samples 3. Tissue homogenization 4. Eicosanoids Extraction 4. Organic solvent extraction 4. Extraction using an octadecylsilyl silica column 5. Chromatographic Separation of Eicosanoids 5. Thin-layer chromatography 5.
Silicic acid column chromatography 5. High-pressure liquid chromatography 6. Quantitative Analysis of Eicosanoids 6. Radioimmunoassay 6. Enzyme immunoassay 6. DuBois These bioactive lipids play an important role in regulating cell proliferation, apoptosis, tissue repair, blood clotting, blood vessel permeability, inflammation, and immune cell behavior. Moreover, some of these eicosanoids also modulate inflammation and tumor growth in cancer tissues, and may serve as biomarkers for monitoring colorectal cancer progression or as an intermediate marker for the pharmacologic activity of chemopreventive agents.
Development of sensitive, rapid, and specific methods for determining eicosanoid levels accurately will facilitate an understanding of the biologic functions of these lipid mediators and will broaden our insight of the importance of these bioactive lipids in vivo.
However, quantitative determination of eicosanoids in biological samples has presented a problem to many investigators. It is necessary to understand the advantages and limitations of each method for quantitative analysis of specific eicosanoids in various types of biological samples. Here we evaluate the methodology of the measurement of eicosanoids in biological samples. Introduction Eicosanoids derived from the arachidonic acid are bioactive lipids that play an important role in the pathogenesis of a variety of human diseases, including inflammation and cancer.
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Arachidonic acid AA is a polyunsaturated fatty acid that constitutes the phospholipid domain of most cell membranes. When tissues are exposed to diverse physiologic and pathologic stimuli, arachidonic acid is liberated from membrane phospholipids by action of cytoplasmic phospholipase A2. Arachidonic acid can be metabolized to eicosanoids through three major pathways: the cyclooxygenase COX pathway, the lipoxygenase LOX pathway, and the cytochrome P monooxygenase pathway. Clinical and epidemiologic studies have already demonstrated the importance of COX and LOX in the progression of colorectal cancer.
Moreover, both pharmacologic and genetic evidence show that some of eicosanoids regulate colorectal tumor growth and are involved in other types of cancer as well Hussey and Tisdale, ; Hussey et al. Therefore, these bioactive lipids may serve as a biomarker for monitoring cancer progression and a valuable intermediate marker for the pharmacological activity of chemopreventive agents.
Successful quantitation of eicosanoids in biological samples is challenging to many investigators. The key regulatory step in this process is the enzymatic conversion of the AA to PGG2, which is then reduced to an unstable endoperoxide intermediate, PGH2. These bioactive lipids exert their cellular functions by binding cell surface receptors that belong to the family of seven transmembrane G-protein—coupled rhodopsin-type receptors. DuBois Wang et al. COX-1—derived PGs play an important role in protecting the gastroduodenal mucosa from damage.
In addition, PGI2 appears to serve an important role in protecting cardiomyocytes from oxidant stress Adderley and Fitzgerald, In contrast, COX-2 derived PGs not only mediate acute inflammatory responses such as swelling, pain, and fever, but are also involved in a variety of pathophysiological processes, including colorectal cancer, ulceration, thrombosis, and kidney disease.
The central role of PGE2 in colorectal tumorigenesis has been further confirmed by evaluating mice with homozygous deletion of PGE2 receptors Mutoh et al. In addition, PGE2 is also a key mediator of acute inflammatory responses Needleman and Isakson, ; Portanova et al. In an eicosanoidgeneration pathway, arachidonic acid was found to serve as a substrate for 5-, 8-, , and LOX which catalyze the stereospecific oxygenation oxidation of the 5-, 8-, , or carbon atoms of arachidonic acid to generate the corresponding hydroperoxyeicosatetraenoic acids HPETEs , respectively.
Several LOXs and their metabolites of arachidonic acid appear to be involved in colon carcinogenesis. In mammals, 14 families and 26 subfamilies of cytochromes P CYPs have been identified. The expression of cytochrome Ps has been identified in a wide range of human cancers Patterson and Murray, For example, CYP1B1 is elevated in tumors including lung, breast, liver, gastrointestinal tract, prostate, and bladder cancers.
The major cytochrome Pgenerated metabolites are the epoxyeicosatrienoic 32 Dingzhi Wang and Raymond N. These products play an important role in the regulation of inflammation, cell migration, apoptosis, and platelet aggregation Capdevila et al. Quantitative Measurement of Eicosanoids It is critical to evaluate the methodology used for the measurement of eicosanoids, particularly for PGE2, because its concentration in the supernatant of colorectal cancer cell lines and in intestinal tissues, plasma, and urine from both humans and mice may serve as a biomarker for monitoring colorectal cancer progression and a valuable intermediate marker for the pharmacological activity of chemopreventive agents.
Therefore, the direct quantitation of PGs is an unreliable indicator in an in vivo system. For this reason, blood, urine, colorectal tissue, or other samples from humans and animals often contain very few intact PGs, and measurement of PG metabolites is crucial to provide a reliable estimate of actual PGs production in the samples that have undergone extensive metabolism before collection Catella et al. The spectrophotometric method is relatively specific and simple, but has serious sensitivity limitations, which is limited to the low microgram range Bygdeman and Samuelsson, ; Shaw and Ramwell, However, these assays have two limitations for tissue and plasma samples.
First, plasma proteins can bind to eicosanoids, which diminishes immunoassay sensitivity. Second, there is a significant degree of immunological cross-reactivity among commercially available eicosanoid antibodies. To solve these problems, an organic solvent or a solid-phase extraction procedure is used to separate eicosanoids from plasma proteins, and chromatographic separation of eicosanoids is employed to avoid immunological cross-reactivity Jaffe et al. In addition, these methods are very useful for the measurement of products in the eicosanoid degradation pathway Green et al.
Preparation of Samples The amount of sample depends on the nature of the sample matrix and the relatively minimal levels of eicosanoids produced by isolated cell suspensions to more complex matrixes such as intestinal tissues, urine, and blood. Sample preparation also depends on the method used for quantitative 34 Dingzhi Wang and Raymond N.
DuBois analysis of eicosanoids. Polypropylene tubes are used throughout the following process to avoid binding of the eicosanoids to glass surfaces. Urine samples In general, 1 to 3 ml of urine is collected for a single measurement. Indomethacin should be added immediately to urine samples at a 10 mM final concentration. Indomethacin will prevent ex vivo formation of eicosanoids. The samples are acidified to pH 3 by adding glacial acetic acid. Organic acids are suggested for this purpose since mineral acids promote the dehydration of PGE2 to PGA2 at a faster rate. Plasma preparation In general, 1 to 2 ml of blood is collected in polypropylene tube coated with a spray-dried anticoagulant such as sodium heparin using plastic syringes.
Indomethacin should be added immediately to blood samples as described previously. Collection of intestinal tumor samples Tumor and matched normal tissue samples range, to mg are collected from human colon or mouse intestines and immediately snap frozen in liquid nitrogen with approximately the same time period between biopsy removal and freezing for each biopsy. All tissue samples should be stored in liquid nitrogen until the time of extraction.
Tissue homogenization Frozen tissue 50 to mg is ground to a fine powder using a liquidnitrogen—cooled mortar Fisher and homogenized in five volumes of icecold PBS buffer with 0. The mixtures are centrifuged at g for 10 min, and the supernatants were transferred to a fresh tube. Eicosanoids Extraction Eicosanoids can be extracted from biological samples using organic solvent or octadecylsilyl ODS silica. The most common method for extracting eicosanoids and eicosanoid metabolites from acidified aqueous solutions is an organic solvent extraction.
However, extraction on a solidphase, such as ODS silica, column is a simple chromatographic method with relative selection and small volumes of the extraction Powell, Organic solvent extraction Ten thousand counts per minute cpm of tritium-labeled eicosanoids of interest are added to the samples 1 to 5 ml of urine or cell-free tissue culture medium, 1 to 3 ml of plasma, or 1 to 5 ml of the supernatant of tissue homogenate , acidified to pH 3 with glacial acetic acid and the lipids are extracted twice with five volumes of diethyl ether and once with five volumes of ethyl acetate.
The organic phase is evaporated to dryness under a stream of nitrogen at room temperature. The dry extract is reconstituted in an appropriate assay buffer for the following procedures. The acidified samples with 10, cpm of tritium-labeled eicosanoids of interest are passed through the prepared cartridge or column using a syringe or under gentle vacuum with a Vac-Elut Vanian Sample Preparation Products, Harbor City, CA. For larger samples incubation volumes up to 25 ml , it may be advisable to use more than one ODS silica cartridge or an open column of ODS silica.
Body fluids such as plasma and urine can 36 Dingzhi Wang and Raymond N. DuBois be applied directly to the prepared cartridge or column after acidification. Eicosanoids can be eluted from ODS silica with organic solvents such as diethyl ether, ethyl acetate, and methyl formate in a siliconized glass or polypropylene tube. Since methyl formate is more polar than diethyl ether and ethyl acetate, only 10 ml are required to elute eicosanoids from the stationary phase. Larger volumes e. The dry extract is reconstituted in an appropriate assay buffer. The column can be reused up to 10 times when extracting culture media and two to three times when extracting urine without contamination or a decrease in extraction efficiency, respectively.
For extracting plasma, the column can be used only once. Chromatographic Separation of Eicosanoids Thin-layer chromatography TLC is the most commonly used technique for the separation of arachidonic acid AA metabolites. However, this method has low and variable recovery and requires the use of several solvent systems to adequately separate the major AA metabolites. Silicic acid column chromatography is a simple chromatographic method but suffers from poor resolution. High-pressure liquid chromatography HPLC offers the advantage of high resolution and good reproducibility. Thin-layer chromatography Unmodified thin-layer silica-coated plates have been used to separate PGs Amin, ; Eastman and Dowsett, ; Green and Samuelsson, Recently, thin-layer silica plates coated with phenylmethylvinylchlorosilane have been employed to separate PGs and HETEs Beneytout et al.
One sample is spotted per lane. The standards 5 mg of each PG are spotted in the outer lanes. A lane is skipped between the standards and the samples to avoid contamination. Samples are spotted next to each other on the inner lanes. The plate is developed with a solution of ethyl acetate-benzene-acetic acid for 30 to 40 min. The plates are allowed to dry for 5 to 10 min and placed in a chamber containing crystals of iodine until the PG standards can be visualized.
The zones of migration of the unknown samples are marked lightly on the silica gel with a pencil. Glass wool plugs are placed in the bottom of Pasteur pipettes. A pipette is placed tip first into a rubber hose that is connected to a vacuum. The zone corresponding to the PG of interest is scraped with a metal spatula. The silica gel is vacuumed into the pipette as it is scraped. After removing all of the loose silica gel, the pipette is removed from the vacuum line and placed tip down in a labeled ml polypropylene tube. This process is repeated for each PG zone of interest.
The PGs are eluted from the silica gel by adding two 2-ml aliquots of chloroform: methanol to the Pasteur pipette. The sample is reconstituted in an appropriate assay buffer depending on the method of quantitative analysis. The recovery of each PG is determined by the scintillation counting. The plate is developed with a solution of heptane:methyl formate:diethyl ether:acetic acid for HETEs.
The distance traveled by solvent is 8. PGs and thromboxane are separated after two migrations heptane:methyl formate:diethyl ether:acetic acid, and hexane:methyl formate:diethyl ether:acetic acid, The distance traveled by first migration is 8. The samples are recovered from the plate as described previously. The silicic acid is pipetted into the column to a level of mm and the column is sequentially rinsed with 6 ml of solvent 1, 38 Dingzhi Wang and Raymond N.
DuBois 6 ml of solvent 2 toluene:ethyl acetate:methanol:water, , and 4 ml of solvent 1. The dry sample extract is dissolved in 0. The combined organic extracts are then applied to the columns. Prostaglandin fractions are obtained by eluting the columns serially with 10 ml of solvent 1 fraction I, PGA2 and PGB2 , 12 ml of solvent 4 toluene: ethyl acetate: methanol: water, 4. High-pressure liquid chromatography Various reverse phase columns such as the Ultrasphere ODS column 5 mm, 4.
The sample with hot tracer and appropriate standards are usually dissolved in 50 to ml of acetone and are injected into the column. Tritiated eicosanoids of interest are run in parallel as standards and used as references for the determination of retention times. For example, the elution times of the eicosanoids are calculated from the [3H] eicosanoid standards.
The column eluate is collected in 0. The detection wavelength is nm. The column eluate is collected in fractions corresponding to the elution times of the [3H] PGs of interest. The flow rate is 1. The column fractions are frozen and lyophilized in a SpeedVac centrifuge Savant. Alternatively, the eicosanoids from the column fractions can also be extracted by organic solvent or solid-phase extraction procedure as described previously. The dried fractions are redissolved in an appropriate assay buffer based on the subsequent method of eicosanoid analysis employed Eling et al.
Quantitative Analysis of Eicosanoids Quantitative determination of eicosanoids can be challenging for some investigators. RIA is rapid and possesses the sensitivity and specificity with lower costs. Similarly, EIA is sensitive and specific, but the cost is much higher. However, antibodies to all the known AA metabolites are not readily available, which limits these techniques. Therefore, these types of analysis methods are restricted to a few research laboratories. Radioimmunoassay Radioimmunoassay RIA has been used to measure the levels of eicosanoids in biological samples. The assay is based on the competition of antigen the eicosanoid being measured with a constant amount of radioactive homologous antigen for a limited amount of specific eicosanoid antibody.
Thus, the necessary reagents are a monospecific antibody, standard unlabeled eicosanoid, and radioactive eicosanoid. In addition, a method of separating antibody-antigen complex from free antigen is required. For more detailed information on the analysis of eicosanoids by RIA, the reader is directed to several excellent reviews Campbell and Ojeda, ; Hollenberg et al. In general, biological samples are extracted by mixing with an organic solvent or solid-phase extraction and purified by reverse-phase HPLC or TLC as described previously.
A standard curve is included in each assay of eicosanoid in duplicate.
Fifty microliters of each appropriately diluted antibody in RIA buffer 0. DuBois and stabilize the separation procedure if added earlier, gelatinization prevents adequate reaction. The simplest and cheapest method of separating bound from free antigen is the adsorption of the free eicosanoid on dextrancoated charcoal DCC.
First, 1. Each eicosanoid standard curve is calculated by plotting the percentage of [3H] eicosanoid bound to the antibody versus picograms of added eicosanoid standard. The concentration of eicosanoid in biological samples is determined by plotting the percentage bound of unknown sample against the standard curve. Recently, RIA kits for several eicosanoids have become commercially available. Enzyme immunoassay Similar to RIA, this assay is also based on the competition between an eicosanoid and a constant amount of chemiluminescent e.
The fraction corresponding to each eicosanoid is collected after purification as described previously and measured by EIA. All standards and samples should be run in duplicate. After another incubation, the enzyme reaction is stopped, and the yellow color generated is read on a microplate reader at a wavelength of nm for acetylcholinesterase reaction and nm for alkaline phosphatase reaction. The intensity of the bound yellow color is inversely proportional to the concentration of the eicosanoid in either standards or samples.
Each eicosanoid standard curve is obtained by plotting percentage bound versus concentration of added eicosanoid standard. The concentration of eicosanoid in biological samples is determined by interpolation. Because EIA kits for most eicosanoids are now commercially available, the detailed procedures for measurement of each eicosanoid are provided by the manufacturers. Ann Arbor, MI. In addition, Assay Designs Inc. Gas chromatography-mass spectrometry Electron-capture, negative-ion, chemical ionization mass spectrometry NCI-MS has been used for the quantitative analysis of eicosanoids in a precise, accurate, and sensitive manner.
An important requirement for this assay is the availability of appropriate stable isotope analogues, which are used as internal standards. The high sensitivity of NCI-MS increases the risk of interfering substances from the biological matrix eluting at the same GC retention time as target eicosanoid.
Such interference can be minimized by the use of suitable purification techniques as describe previously. All eicosanoids and their related deuterated standards can be purchased from Cayman Chemical Co. The pentafluorobenzyl PFB ester is the most widely used electron-capturing derivative for eicosanoids. However, eicosanoids often contain polar hydroxyl groups that cause tailing during analysis by gas chromatography GC.
This problem can be overcome by conversion of the hydroxyl groups to trimethylsilyl TMS ethers. However, a ketone group in some eicosanoids can form unstable enol-TMS derivatives. This problem can be solved by the conversion of the ketone to a methoxime MO derivative using O-methoxylamine HCl before any derivatization. DuBois 6. Analysis of prostanoids The dried PGs are supplemented with 25 ml deuterated PG standards 3 ng for each and ml acetone, and the combined solution is dried under a steady stream of N2.
The reaction mixtures are then dried under nitrogen, and this procedure is repeated to ensure quantitative PFB esterification. After the second esterification, the reaction mixtures are dried under N2 and the residue is subjected to TLC using the solvent system ethyl acetate:methanol Approximately 2 to 5 mg of the pentafluorobenzyl ester of each PG standard is subjected to TLC on a separate lane. Corresponding zones containing the prostanoids in the unknown samples are scraped and the compounds are eluted with methanol as described previously.
The reaction mixtures are then dried under nitrogen and the residue is redissolved in 10 ml of undecane. Gas chromatographic analyses are carried out on a DB or DB-1 fused silica capillary column 15 m, 0. Standard curves for prostanoids are prepared by addition of each prostanoid 0. The ratio of peak areas is plotted against the amount of each prostanoid added, which results in a linear relation. The amount of endogenous prostanoids is determined by plotting the ratio of peak areas of unknown sample against the standard curve. After derivatization, the samples are dissolved in decane and analyzed by GC-MS.
Helium is used as a carrier gas with a linear velocity of 0. Electron-capture ionization is carried out using methane as a moderating gas at a flow resulting in ion source pressure of approximately 1. The hydrolysis mixture is acidified to pH 1. After derivatization by pentafluorobenzylbromide and bis trimethylsilyl trifluoroacetamide, samples are analyzed by GC-MS as described previously.
Each sample 1. The mass spectrometer operating conditions are typically accelerating voltage, 8 kV; ionization energy, 80 eV; emission current, 1 mA; and resolution, A standard curve is constructed by adding a constant amount of [2H4] LTB4 10 ng, 2. The amount of LTB4 in the unknown samples is determined as described previously. Liquid chromatography-mass spectrometry-mass spectrometry Analysis of eicosanoids by GC-MS method is time consuming and complicated. Recently, LC-MS-MS has been used to simultaneously quantify endogenous eicosanoids in in vitro and in vivo samples without purification procedures Kempen et al.
Before extraction, each sample collected from cell-free tissue culture medium, urine, plasma, or tissue homogenate is mixed with a mixture of known quantities of deuterated eicosanoids of interest 2 ng for each deuterated eicosanoid. The samples are extracted by organic solvent as described previously, and the dried extract is reconstituted in ml of methanol:ammonium acetate buffer 10 mM at pH 8.
Twenty-five microliters of sample are injected into the column. The mobile phase consists of mM ammonium acetate pH 8. Fragmentation for all compounds is performed using argon as the collision gas at a collision cell pressure of 2. The collision energy is 19 V. All eicosanoids are detected using electrospray negative-ionization and multiple-reaction monitoring of the transition ions for the metabolites and their internal standards. Samples are then extracted by a C18 SepPak and dried under N2 as described previously.
The dry extracts are resuspended in ml mobile phase A 5-mM ammonium acetate:acetonitrile:acetic acid, We also thank the T. Amin, A. Nitric oxide synthase and cyclooxygenases: Distribution, regulation, and intervention in arthritis. Amin, M. Direct quantitative thin-layer chromatographic determination of prostaglandins A2, B2, E2 and F2 alpha.
Anderson, G. Selective inhibition of cyclooxygenase COX -2 reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis. Beneytout, J. Separation of arachiclonic acid metabolites by thin-layer chromatography using new silicone-bonded plates.
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High Res. Bhatia, B. Subcellular localization and tumor-suppressive functions of lipoxygenase 2 LOX2 and its splice variants. DuBois Bortuzzo, C. Brouard, C. Effects of moderate dietary supplementations with n-3 fatty acids on macrophage and lymphocyte phospholipids and macrophage eicosanoid synthesis in the rat. Acta , 19— Bygdeman, M. Quantitative determination of prostaglandins in human semen. Acta 10, — Campbell, W. Measurement of prostaglandins by radioimmunoassay. Methods Enzymol. Capdevila, J. Cytochrome P and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase.
Catella, F. Measurement of renal and non-renal eicosanoid synthesis. Cellai, I. Antineoplastic effects of rosiglitazone and PPARgamma transactivation in neuroblastoma cells. Cancer 95, — Chen, J. Epoxyeicosatrienoic acids and their sulfonimide derivatives stimulate tyrosine phosphorylation and induce mitogenesis in renal epithelial cells. Transfection of an active cytochrome P arachidonic acid epoxygenase indicates that 14,epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor.
Craven, P. Patterns of prostaglandin synthesis and degradation in isolated superficial and proliferative colonic epithelial cells compared to residual colon. Prostaglandins 26, — Del Vecchio, R. A quantitative solid-phase enzymeimmunoassay for 13,dihydroketo-prostaglandin F2 alpha in plasma. Prostaglandins 43, — DuBois, R. Cyclooxygenase in biology and disease. Eastman, A. The simultaneous separation of individual prostaglandins by thin-layer chromatography on an unmodified support. Eling, T. Separation of arachidonic acid metabolites by high-pressure liquid chromatography.
Ensor, C. Fleming, I. Endothelium-derived hyperpolarizing factor synthase cytochrome P 2C9 is a functionally significant source of reactive oxygen species in coronary arteries. Forman, B. Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta. USA 94, — Cell 83, — Measurement of icosanoids. Prostaglandins 27, — Giardiello, F. Prostanoids, ornithine decarboxylase, and polyamines in primary chemoprevention of familial adenomatous polyposis.
Gastroenterology , — Gould, S. Studies of prostaglandins and sulphasalazine in ulcerative colitis. Prostaglandins Med. Green, K. Methods for quantitative analysis of PGF2, PGE2, 9, 11 -dihydroxyketo-prostenoic acid and 9, 11, trihydroxy-prostenoic acid from body fluids using deuterated carriers and gas chromatography-mass spectrometry. Prostaglandins and related factors. Thin-layer chromatography of prostaglandins. Gupta, R. Prostacyclin-mediated activation of peroxisome proliferatoractivated receptor delta in colorectal cancer.
USA 97, — Activation of nuclear hormone receptor peroxisome proliferator-activated receptor-delta accelerates intestinal adenoma growth. Hansen-Petrik, M. Herschman, H. Prostaglandin synthase 2. Inflammation, reproduction, cancer and all that The regulation and role of the inducible prostaglandin synthase. Bioessays 17, — Hoebel, B. Hollenberg, S. Eicosanoid production by human aortic endothelial cells in response to endothelin. Hussey, H. Pick and Choose. Literature Updates. For Members. For Librarians. RSS Feeds. Chemistry World. Education in Chemistry. Open Access. Historical Collection.
Browning et al. PC is an important phospholipid involved in lipid metabolism. PC has been shown to play a major role in cellular proliferation, degeneration, and membrane fluidity and functions [ 13 ]. The physiological properties of PC are heavily dependent on their fatty acid composition and n -3 PUFA has been shown to be preferentially incorporated into PC [ 48 ]. In addition to their anti-inflammatory effects, n -3 PUFA rich PC are also known to possess lipid lowering effects [ 51 - 53 ]. These bioactive metabolites of PC can also elicit beneficial effects depending on the type, chain length, and degree of unsaturation of their fatty acids.
PC is an important source of FFA in plasma [ 48 ]. This is in line with previous findings that support the incorporation of n -3 PUFA into the plasma pool when given as dietary supplements [ 48 , 54 ]. Our observation is in line with the study of Lamaziere et al. Chronic low-grade inflammation underlies the pathology of most metabolic disorders, and free n -3 PUFA has been shown to possess potent anti-inflammatory properties [ 58 ].
N -3 PUFA alleviates inflammation by directly regulating transcription factors involved in inflammation [ 59 - 61 ] and indirectly by producing series-3 and series-5 eicosanoids [ 62 , 63 ]. In addition to their inflammation resolving properties, free unesterified n -3 PUFA have also been shown to improve symptoms of dyslipidaemia [ 9 - 11 ]. Similar to our plasma data, we found a higher concentration of hepatic LPC and a low concentration of hepatic LPC in the high n -3 group. Interestingly, there was no significant difference in hepatic LPC, although there was a trend towards an increase in the high n-3 PUFA group.
Ottestad et al. Another study by Block et al.
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LPC has been suggested as the major carrier of DHA to the brain tissues [ 66 ]; tracer studies revealed that labelled LPC injected into the blood of rat disappeared within 20 s and were recovered in different organs including the brain [ 17 ]. Studies have controversially linked LPC with the development of atherosclerosis [ 25 - 27 ]. This is possibly due to their association with oxidized LDL, and promotion of inflammation [ 67 , 68 ] by generating reactive oxygen species and nitric oxides in different types of cells [ 69 , 70 ]. However, the studies that linked LPC with the pathogenesis of obesity, diabetes, and rheumatoid arthritis have reported an increase in saturated LPC [ 30 ].
Of paramount importance to the biological functions of LPC are acyl length and degree of saturation of their fatty acids [ 65 ]. We have not investigated the position of the acyl group of our LPC, however, it is known that there is rapid isomerization of acyl group from sn -2 to a more stable sn -1 position in LPC [ 75 ]. Also noteworthy is the fact that, despite the changes in fatty acyl species of PC and LPC in response to diet, there was no difference in the total concentrations of PC and LPC between the two experimental groups.
A similar observation was found by Ottestad et al. This interesting observation suggests that the functional properties of n -3 PUFA involve remodelling and improving the quality of bioactive lipid mediators without affecting their concentrations. CE is less polar than free cholesterol and it functions as an inert storage molecule. This would simply indicate that n -3 PUFA are stored and will be later released for other physiological functions.
There was no difference in plasma PE between the two dietary groups; the only difference detected in the liver was an increase in PE in the high n -3 group. CER is a sphingolipid linked with inflammation and the pathogenesis of cardiovascular diseases CVD [ 77 , 78 ]. There are also speculations on the involvement of CER in insulin signalling, although available information is scanty [ 79 , 80 ].
We found no difference in total concentration of CER in plasma and liver. Our findings are similar to Ottestad et al. The functional roles of this CER species are currently unknown and needs to be explored. There was no difference in plasma and liver SM concentrations between the two groups.
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The physiological functions of SM have not been extensively studied. However, it is known that SM are involved in the formation of specialized membrane microdomains known as lipid rafts involved in signalling. In conclusion, our findings have shown that diets high in n -3 PUFA alter plasma and liver lipidomic profile of the offspring. We found that n -3 PUFA is preferentially incorporated into PC and LPC, and despite the changes in lipidomic profile, the total concentrations of these lipids were not altered.
Additionally, we found that dietary n -3 PUFA is capable of remodelling the fatty acyl moieties of PC, LPC, and CE, which may have important physiological implications, and needs to be further investigated. Future studies will be undertaken to investigate the mechanism s by which n -3 PUFA remodelled bioactive lipids regulate metabolic pathways. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Abstract Background Omega n -3 polyunsaturated fatty acids PUFA are converted to bioactive lipid components that are important mediators in metabolic and physiological pathways; however, which bioactive compounds are metabolically active, and their mechanisms of action are still not clear. Introduction Essential polyunsaturated fatty acids PUFA of the omega-3 n -3 and n -6 classes are important in the regulation of metabolic processes. Fatty Acid High n -3 Low n -3 C 1. Lipids were extracted from the diets and the fatty acid composition was determined by gas chromatography.
Lipidomic analysis Lipid extraction, standards, and solvents. Statistical analysis Data were analysed using GraphPad Prism software version 5. Download: PPT. Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Discussion N -3 PUFA have been shown to reduce plasma TG [ 8 , 45 ], prevent atherosclerosis [ 46 ], and alleviate inflammation [ 47 ]. Supporting Information. File S1. References 1.
Calder PC n-3 Fatty acids and cardiovascular disease: evidence explained and mechanisms explored. Clin Sci Lond PubMed: View Article Google Scholar 2.