Electrospray ionization tandem mass spectrometric study of the aconitines in the roots of aconite

Fragmentation pathways of aconitine-type alkaloids were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry. Low-energy collision-induced dissociation of protonated aconitines follows a dominant first step, the elimination of the C-8-substituent as acetic acid or fatty acid in MS2 spectra. Successive losses of 1-4 CH3OH molecules, 1-3 H2O, CO, benzoic acid, and CH3 or C2H5 (N-substituents) are all fragmentation pathways observed in MS3 and MS4 spectra. By applying knowledge of these fragmentation pathways to the aconitines in the ethanolic extract of aconite roots, all the known aconitines were detected and also 23 unknown aconitine-type alkaloids, in which the lipo-alkaloids containing residues of 15C, 17C and 19C saturated or unsaturated fatty acids were characterized. These odd-carbon-number fatty acid substituents have not been reported previously.

Positional distribution of fatty acids on the glycerol backbone during the biosynthesis of glycerolipids in Ectocarpus fasciculatus

The biosynthesis of glycolipids in E. fasciculatus was studied by C-14 label and chase. The fatty acids in sulphoquinovosyl diacylglycerol (SQDG) were almost 16-carbon and 18-carbon ones. In addition to the two fatty acids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) contained 8.5 mol% and 31.0 mol% of eicosapentaenoic acid (20 : 5), respectively, and this fatty acid was usually distributed in the sn-1 position of the glycerol backbone. When plants were incubated with [2-C-14] acetate, differences existed in the positional distribution of the labeled fatty acids in sn-1 and sn-2 among the three glycerolipids. In SQDG C-14-labeled fatty acids were distributed uniformly in the sn-1 and sn-2 positions. In DGDG, C-14-labeled fatty acids were mainly distributed in the sn-2 position. In MGDG, the radioactivity of fatty acids in sn-1 position was far greater than that in sn-2 position after a 30 min pulse label, and the difference in radioactivity between the two positions decreased rapidly. The above results indicated that differences in the positional distribution of C-14-labeled fatty acids between sn-1 and sn-2 positions might be related to 20 : 5 and the biosynthesis of DGDG. Our results also suggested that E. fasciculatus had the same DGDG biosynthetic pathway as that in higher plants and galactosyl transferase was selective for MGDC.

Effect of alginic acid decomposing bacterium on the growth of Laminaria japonica (Phaeophyceae)

We collected the diseased blades of Laminaria japonica from Yantai Sea Farm from October to December 2002, and the alginic acid decomposing bacterium on the diseased blade was isolated and purified, and was identified as Alterornonas espejiana. This bacterium was applied as the causative pathogen to infect the blades of L. japonica under laboratory conditions. The aim of the present study was to identify the effects of the bacterium on the growth of L. japonica, and to find the possibly effective mechanism. Results showed that: (1) The blades of L. japonica exhibited symptoms of lesion, bleaching and deterioration when infected by the bacterium, and their growth and photosynthesis were dramatically suppressed. At the same time, the reactive oxygen species (ROS) generation enhanced obviously, and the relative membrane permeability increased significantly. The contents of malonaldehyde (MDA) and free fatty acid in the microsomol membrane greatly elevated, but the phospholipid content decreased. Result suggested an obvious peroxidation and deesterrification in the blades of L. japonica when infected by the bacterium. (2) The simultaneous assay on the antioxidant enzyme activities demonstrated that superoxide dismutase (SOD) and catalase (CAT) increased greatly when infected by the bacterium, but glutathione peroxidase (Gpx) and ascorbate peroxidase (APX) did not exhibit active responses to the bacterium throughout the experiment. (3) The histomorphological observations gave a distinctive evidence of the severity of the lesions as well as the relative abundance in the bacterial population on the blades after infection. The bacterium firstly invaded into the endodermis of L. japonica and gathered around there, and then resulted in the membrane damage, cells corruption and ultimately, the death of L. japonica.

Fatty acids of some algae from the Bohai Sea

The fatty acid compositions of 22 species of marine macrophytes, belonging to the Ceramiales, Cryptonemiales, Nemalionales, Laminariales, Chordariales, Scytosiphonales, Desmarestiales, Dictyosiphonales, Fucales, Dictyotales and Ulvales and collected from the Bohai Sea, were determined by capillary gas chromatography. The contents of polyunsaturated fatty acids (FAs) in the Bohai Sea algae, in comparison with the same species from the Yellow Sea were found to be lower. Red algae had relatively high levels of the acids 16:0, 18:1(n-7), 18:1(n-9), 20:5(n-3) and 20:4(n-6), and those examined were rich in C-20 PUFAs, these chiefly being arachidonic and eicosapentaenoic acids. The major FAs encountered in the Phaeophyta were 14:0, 16:0, 18:1(n-9), 18:2(n-6), 18:3(n-3), 18:4(n-3), 20:4(n-6) and 20:5(n-3). C18PUFAs are of greater abundance in the brown algae than in the red algae examined. All three green algae from the Ulvales had similar fatty acid patterns with major components, 16:0, 16:4(n-3), 18:1(n-7), 18:2(n-6), 18:3(n-3), and 18:4(n-3). They contained 16:3(n-3) and more 16:4(n-3), were rich in C18PUFAs, chiefly 18:3(n-3) and 18:4(n-3) and had 18:1(n-7)/18:1 (n-9) ratios higher than 1. (C) 2002 Elsevier Science Ltd. All rights reserved.

Necessity of dietary lecithin and eicosapentaenoic acid for growth, survival, stress resistance and lipoprotein formation in gilthead sea bream Sparus aurata

The substitution of dietary docosahexaenoic acid (DHA) with eicosapentaenoic acid (EPA) reduces larval growth in gilthead sea bream. However, the value of EPA when dietary DHA is able to meet the requirements of the larvae has not been sufficiently studied. Dietary phosphoacylgliceride levels also affect fish growth and it has been suggested that they enhance lipid transport in developing larvae. The present experiment was carried out to further study the effect of dietary lecithin and eicosapentaenoic acid on growth, survival, stress resistance,. larval fatty acid composition and lipid transport, when DHA is present in the microdiets of gilthead:sea bream. Eighteen thousand gilt-head sea bream larvae of 4.99+/-0.53 mm total length were fed three microdiets tested by triplicate: a control diet [2% soybean lecithin (SBL) and 2.89% EPA], a low EPA diet,(2% SBL and 1.63% EPA) and a no SBL diet (0% SBL and 2.71% EPA). Handling, temperature and salinity tests determined larval resistance to stress. The results show that when dietary DHA levels are high, but dietary arachidonic acid (ARA) levels are about 0.2%, EPA is necessary to improve larval growth, and survival. Larval EPA content, but not DHA or ARA, was affected by dietary EPA levels. Increased dietary EPA improved larval stress resistance to handling and temperature tests, which could be related to its possible role as a regulator of cortisol production whereas it did not affect stress resistance after salinity shock. Larvae fed the no SBL diet showed a lower lipid content characterized by a low proportion of saturated and monounsaturated fatty acids, together with a significant reduction in the appearance of lipoprotein particles in the lamina propria and in the size of such particles, denoting a critical reduction in dietary lipid transport and utilization, and lower larval growth and survival rates.

Extraction of Nitraria tangutorum seed oil by supercritical carbon dioxide and determination of free fatty acids by HPLC/APC/IMS with fluorescence detection

The seed oil from Nitraria tangutorum samples was obtained by supercritical carbon dioxide extraction methods. The extraction parameters for this methodology, including pressure, temperature, particle size and extraction time, were optimized. The free fatty acids in the seed oil were separated with a pre-column derivation method and 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-p-toluenesulfonate (BDETS) as a labeling regent, followed by high-performance liquid chromatography (HPLC) with fluorescence detection. The target compounds were identified by mass spectrometry with atmospheric pressure chemical ionization (APCI in positive-ion mode). HPLC analysis shows that the main compositions of the seed oil samples were free fatty acids (FFAs) in high to low concentrations as follows: linoleic acid, oleic acid, hexadecanoic acid and octadecanoic acid. The assay detection limits (at signal-to-noise of 3:1) were 3.378-6.572 nmol/L. Excellent linear responses were observed, with correlation coefficients greater than 0.999. The facile BDETS derivatization coupled with mass spectrometry detection allowed the development of a highly sensitive method for analyzing free fatty acids in seed oil by supercritical CO2 extraction. The established method is highly efficient for seed oil extraction and extremely sensitive for fatty acid profile determination. (C) 2007 Elsevier B.V. All rights reserved.

Determination of free fatty acids in soil and bryophyte plants by precolumn derivatization via HPLC with fluorescence detection and tandem mass spectrometry (HPLC-MS/MS)

A sensitive method for the determination of 30 kinds of free fatty acids (FFAs, C-1-C-30) with 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f] 9,10-phenan- threne (TSPP) as labeling reagent and using high performance liquid chromatography with fluorescence detection and identification by online postcolumn mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion mode (HPLC/MS/APCI) has been developed. TSPP could easily and quickly label FFAs in the presence of K2CO3 catalyst at 90 degrees C for 30 min in N,N-dimethylformamide (DMF) solvent, and maximal labeling yields close to 100% were observed with a 5-fold excess of molar reagent. Derivatives were stable enough to be efficiently analyzed by high performance liquid chromatography. TSPP was introduced into fatty acid molecules and effectively augmented MS ionization of fatty acid derivatives and led to regular MS and MS/MS information. The collision induced cleavage of protonated molecular ions formed specific fragment ions at m/z [MH](+)(molecular ion), m/z [M'+CH2CH2](+)(M' was molecular mass of the corresponding FFA) and m/z 295.0 (the, mass of protonated molecular core structure of TSPP). Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C-8 column (4.6 x 150 mm, 5 mu m, Agilent) with a good baseline resolution in combination with a gradient elution. Linear ranges of 30 FFAs are 2.441 x 10(-3) to 20 mu mol/L, detection limits are 3.24 similar to 36.97 fmol (injection volume 10 mu L, at a signal-to-noise ratio of 3, S/N 3:1). The mean interday precision ranged from 93.4 to 106.2% with the largest mean coefficients of variation (R.S.D.) < 7,5%. The mean intraday precision for all standards was < 6.4% of the expected concentration. Excellent linear responses were observed with correlation coefficients of > 0.9991. Good compositional data could be obtained from the analysis of extracted fatty acids from as little as 200 mg of bryophyte plant samples.Therefore, the facile TSPP derivatization coupled with HPLC/MS/APCI analysis allowed the development of a highly sensitive method for the quantitation of trace levels of short and long chain fatty acids from biological and natural environmental samples.

Determination of 30 free fatty acids in two famous Tibetan medicines by HPLC with fluorescence detection and mass spectrometric identification

A simple and sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection and mass spectrometric identification has been developed for analysis of 30 long-chain and short-chain free Fatty acids (FFAs). The fatty acids were derivatized to their esters with 1-[2-(p-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) in N,N-dimethylformamide (DMF) at 90 degrees C with anhydrous K2CO3 as catalyst. A mixture Of C-1-C-30 fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C-8 column. Qualitative identification of the acids was performed by atmospheric-pressure chemical ionization mass spectrometry (APCI-MS) in positive-ion mode. The fluorescence excitation and emission wavelengths were 260 and 380 nm, respectively. Quantitative determination of the 30 acids in two Tibetan medicines Gentiana straminea and G. dahurica was performed. The results indicated that the medicines contained many FFAs. Linear correlation coefficients for the FFA derivatives were > 0.9991. Relative standard deviations (RSDs, n = 6) for the fatty acid derivatives were < 3%. Detection limits (at a signal-to-noise ratio of 3:1) were 3.1-38 fmol. When the fatty acid derivatives were determined in the two real samples results were satisfactory and the sensitivity and reproducibility of the method were good.

Analysis of saturated free fatty acids from pollen by HPLC with fluorescence detection

A sensitive method for the determination of free fatty acids using 2-(2-(anthracen-10-yl)-1H-naphtho[2,3-dimidazol-1-yl) ethyl-p-toluenesuIfonate (ANITS) as tagging reagent with fluorescence detection has been developed. ANITS could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90 degrees C for 40 min in N,N-dimethylformamide solvent. From the extracts of rape bee pollen samples, 20 free fatty acids were sensitively determined. Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C8 column by HPLC in conjunction with gradient elution. The corresponding derivatives were identified by post-column APCI/MS in positive-ion detection mode. ANITS-fatty acid derivatives gave an intense molecular ion peak at mlz [M+H](+); with MS/MS analysis, the collision-induced dissociation spectra of m/z [M+H](+) produced the specific fragment ions at mlz [M-345](+) and mlz 345.0 (here, m/z 345 is the core structural moiety of the ANITS molecule). The fluorescence excitation and emission wavelengths of the derivatives were lambda(ex) = 250 nm and lambda(em) = 512 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are > 0.9999. Detection limits, at a signal-to-noise ratio of 3 : 1, are 24.76-98.79 fmol for the labeled fatty acids.

HPLC-APCI-MS determination of free fatty acids in Tibet folk medicine Lomatogonium rotatum with fluorescence detection and mass spectrometric identification

A simple and sensitive method for the determination of free fatty acids (FFAs) using acridone-9-ethyl-p-toluenesulfonate (AETS) as a fluorescence derivatization reagent by high performance liquid chromatography (HPLC) has been developed. Free fatty acid derivatives were separated on an Eclipse XDB-C-8 column with a good baseline resolution and detected with the fluorescence of which excitation and emission wavelengths of derivatives were set at lambda(ex) 404 and lambda(em) 440 nm, respectively. Identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with an atmospheric pressure chemical ionization (APCI) source under positive-ion detection mode. Nineteen FFAs from the extract of Lomatogonium rotatum are sensitively determined. The results indicate that the plant Lomatogonium rotatum is enriched with an abundance of FFAs and FFAs of higher contents, which mainly focus on even carbon atoms, C-14, C-16, and C-18. The validation of the method including linearity, repeatability, and detection limits was examined. Most linear correlation coefficients for fatty acid derivatives are > 0.9989, and detection limits (at signal-to-noise of 3: 1) are 12.3-43.7 fmol. The relative standard deviations (RSDs) of the peak areas and retention times for 19 FFAs standards are < 2.24% and 0.45%, respectively. The established method is rapid and reproducible for the separation determination of FFAs from the extract of Lomatogonium rotatum with satisfactory results.

Simple derivatization method for sensitive determination of fatty acids with fluorescence detection by high-performance liquid chromatography using 9-(2-hydroxyethyl)-carbazole as derivatization reagent

A simple and sensitive method for the determination of short and long-chain fatty acids using high-performance liquid chromatography with fluorimetric detection has been developed. The fatty acids were derivatized to their corresponding esters with 9-(2-hydroxyethyl)-carbazole (HEC) in acetonitrile at 60 degreesC with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C-1-C-20 fatty acids was completely separated within 38 min in conjunction with a gradient elution on a reversed-phase C-18 column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (lambda (ex) 335 nm). Studies on derivatization conditions indicate that fatty acids react proceeded rapidly and smoothly with HEC in the presence of EDC and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The R.S.D. (n = 6) for each fatty acid derivative are <4%. The detection limits are at 45-68 fmol levels for C-14-C-20 fatty acids and even lower levels for fatty acids. (C) 2001 Elsevier Science B.V. All rights reserved.

Polyunsaturated eicosapentaenoic acid changes lipid composition in lipid rafts

Background Polyunsaturated fatty acids (PUFAs) modulate immune responses particularly by affecting T cell function and are applied clinically as adjuvant immunosuppressants in the treatment of various inflammatory diseases. However, the molecular mechanisms of PUFA-induced immunosuppressive effects are not yet elucidated. Membrane lipid rafts are functional plasma membrane microdomains characterized by a unique lipid environment. Since lipid interactions are crucial for the formation of lipid rafts, the immunomodulatory effects of PUFAs may be due to changes of fatty acid composition in lipid rafts.

beta-Arrestin1 mediates nicotinic acid-induced flushing, but not its antilipolytic effect, in mice.

Nicotinic acid is one of the most effective agents for both lowering triglycerides and raising HDL. However, the side effect of cutaneous flushing severely limits patient compliance. As nicotinic acid stimulates the GPCR GPR109A and Gi/Go proteins, here we dissected the roles of G proteins and the adaptor proteins, beta-arrestins, in nicotinic acid-induced signaling and physiological responses. In a human cell line-based signaling assay, nicotinic acid stimulation led to pertussis toxin-sensitive lowering of cAMP, recruitment of beta-arrestins to the cell membrane, an activating conformational change in beta-arrestin, and beta-arrestin-dependent signaling to ERK MAPK. In addition, we found that nicotinic acid promoted the binding of beta-arrestin1 to activated cytosolic phospholipase A2 as well as beta-arrestin1-dependent activation of cytosolic phospholipase A2 and release of arachidonate, the precursor of prostaglandin D2 and the vasodilator responsible for the flushing response. Moreover, beta-arrestin1-null mice displayed reduced cutaneous flushing in response to nicotinic acid, although the improvement in serum free fatty acid levels was similar to that observed in wild-type mice. These data suggest that the adverse side effect of cutaneous flushing is mediated by beta-arrestin1, but lowering of serum free fatty acid levels is not. Furthermore, G protein-biased ligands that activate GPR109A in a beta-arrestin-independent fashion may represent an improved therapeutic option for the treatment of dyslipidemia.

Polyunsaturated fatty acids in the pathogenesis and treatment of multiple sclerosis

Epidemiological, biochemical, animal model and clinical trial data described in this overview strongly suggest that polyunsaturated fatty acids, particularly n-6 fatty acids, have a role in the pathogenesis and treatment of multiple sclerosis (MS). Data presented provides further evidence for a disturbance in n-6 fatty acid metabolism in MS. Disturbance of n-6 fatty acid metabolism and dysregulation of cytokines are shown to be linked and a "proof of concept clinical trial" further supports such a hypothesis. In a randomised double-blind, placebo controlled trial of a high dose and low dose selected GLA (18:3n-6)-rich oil and placebo control, the high dose had a marked clinical effect in relapsing-remitting MS, significantly decreasing the relapse rate and the progression of disease. Laboratory findings paralleled clinical changes in the placebo group in that production of mononuclear cell pro-inflammatory cytokines (TNF-alpha, IL-1 beta) was increased and anti-inflammatory TGF-beta markedly decreased with loss of membrane n-6 fatty acids linoleic (18:2n-6) and arachidonic acids (20:4n-6). In contrast there were no such changes in the high dose group. The improvement in disability (Expanded Disability Status Scale) in the high dose suggests there maybe a beneficial effect on neuronal lipids and neural function in MS. Thus disturbed n-6 fatty acid metabolism in MS gives rise to loss of membrane long chain n-6 fatty acids and loss of the anti-inflammatory regulatory cytokine TGF-beta, particularly during the relapse phase, as well as loss of these important neural fatty acids for CNS structure and function and consequent long term neurological deficit in MS.

Towards the Industrial Production of Omega-3 Long Chain Polyunsaturated Fatty Acids from a Genetically Modified Diatom Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum can accumulate up to 30% of the omega-3 long chain polyunsaturated fatty acid (LC-PUFA) eicosapentaenoic acid (EPA) and, as such, is considered a good source for the industrial production of EPA. However, P. tricornutum does not naturally accumulate significant levels of the more valuable omega-3 LC-PUFA docosahexaenoic acid (DHA). Previously, we have engineered P. tricornutum to accumulate elevated levels of DHA and docosapentaenoic acid (DPA) by overexpressing heterologous genes encoding enzyme activities of the LC-PUFA biosynthetic pathway. Here, the transgenic strain Pt_Elo5 has been investigated for the scalable production of EPA and DHA. Studies have been performed at the laboratory scale on the cultures growing in up to 1 L flasks a 3.5 L bubble column, a 550 L closed photobioreactor and a 1250 L raceway pond with artificial illumination. Detailed studies were carried out on the effect of different media, carbon sources and illumination on omega-3 LC-PUFAs production by transgenic strain Pt_Elo5 and wild type P. tricornutum grown in 3.5 L bubble columns. The highest content of DHA (7.5% of total fatty acids, TFA) in transgenic strain was achieved in cultures grown in seawater salts, Instant Ocean (IO), supplemented with F/2 nutrients (F2N) under continuous light. After identifying the optimal conditions for omega-3 LC-PUFA accumulation in the small-scale experiments we compared EPA and DHA levels of the transgenic strain grown in a larger fence-style tubular photobioreactor and a raceway pond. We observed a significant production of DHA over EPA, generating an EPA/DPA/DHA profile of 8.7%/4.5%/12.3% of TFA in cells grown in a photobioreactor, equivalent to 6.4 μg/mg dry weight DHA in a mid-exponentially growing algal culture. Omega-3 LC-PUFAs production in a raceway pond at ambient temperature but supplemented with artificial illumination (110 μmol photons m-2s-1) on a 16:8h light:dark cycle, in natural seawater and F/2 nutrients was 24.8% EPA and 10.3% DHA. Transgenic strain grown in RP produced the highest levels of EPA (12.8%) incorporated in neutral lipids. However, the highest partitioning of DHA in neutral lipids was observed in cultures grown in PBR (7.1%). Our results clearly demonstrate the potential for the development of the transgenic Pt_Elo5 as a platform for the commercial production of EPA and DHA.