R ・ メンツェルとドイツ研究振興協会 (RU RU780/11-1) によって L. ・ ルッスの財政支援は、感謝します。R. Nehring RU 780/10-1 によって資金を供給されました。最後に、原稿の校正のため博士ハシバミ Ruvimbo Maboreke に非常に感謝しています。

<ol>
	<li>Ruess, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+lipid+analysis+to+understand+trophic+interactions+in+soil.">Application of lipid analysis to understand trophic interactions in soil.</a> Ecology. 86 (8), 2075-2082 (2005).</li><li>Ruess, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+composition+of+Collembola+and+their+food+resources+in+deciduous+forest+stands+-+Implications+for+feeding+strategies.">Lipid composition of Collembola and their food resources in deciduous forest stands - Implications for feeding strategies.</a> Soil Biology and Biochemistry. 39 (8), 1990-2000 (1990).</li><li>Chamberlain, P. M., Bull, I. D., Black, H. I. J., Ineson, P., Evershed, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid+composition+and+change+in+Collembola+fed+differing+diets:+identification+of+trophic+biomarkers.">Fatty acid composition and change in Collembola fed differing diets: identification of trophic biomarkers.</a> Soil Biology and Biochemistry. 37 (9), 1608-1624 (2005).</li><li>Stott, A. W., Davies, E., Evershed, R. P., Tuross, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+routing+of+dietary+and+biosynthesised+lipids+through+compound-specific+stable+isotope+(delta+C-13)+measurements+at+natural+abundance.">Monitoring the routing of dietary and biosynthesised lipids through compound-specific stable isotope (delta C-13) measurements at natural abundance.</a> Naturwissenschaften. 84 (2), 82-86 (1997).</li><li>Ruess, L., Haggblom, M. 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Isotopologue のプロファイル
FAs 13C 分布の定量的側面の詳細な分析では、炭素の食物網でのパーティション分割を割り当てるには最先端の技術を必要があります。現在の仕事は isotopologue 13C を評価するためにプロファイリングを採用/熱帯の相互作用のための共通の FA バイオ マーカー12C 比。このメソッドでは、液体クロマトグラフィー (...

土壌など不可解な生息地で栄養関係解決困難な動物のサイズが小さいによってさらに制限されます。最後の十年は、フィールド条件1,2,3の下の土壌動物相の供給戦略を定義するためのバイオ マーカーとしての脂肪酸の使用で特に、生化学的な生態学の進歩を見ています。これは全体の分子として消費者組織のリソースから脂肪酸を組み込むことができるという事実に基づく、食事ルーティング4と呼ばれる、プロセス。脂肪酸の転送は、以上 3 つ栄養のレベル、すなわち、菌類から粘管目5線虫に報告されています。最近では、捕食動物は、6、7を考慮され、土壌食物網における栄養マーカーとして脂肪酸の最初のレビューが公開された8,9をされています。
栄養相互作用の詳細については、脂肪酸の安定同位体プロービング (FA SIP) によって達成されます。13C の定量/ダイエットと消費することができます脂肪酸12C 比バイナリ リンクのせいと関連付けられている炭素フローを見積もるし、地上波で採用されている、淡水、海洋の食物網10,11 ,12,13。基本的な前提は、ルーティングされた脂肪酸が酵素プロセスの対象とはしたがっての13C 信号、すなわち、 13C/12C 消費者の脂肪酸の比はダイエット1に類似しています。ただし、食物連鎖を13C 署名の段階的な減少は栄養研究14,,1516FA SIP の広範なアプリケーションをそれにより妨げる水生システムで報告されています。さらに、地上の食物網におけるほとんどの無脊椎動物の脂質代謝の知識はまだ限られています。
栄養マーカー脂肪酸食物網生態学における定量的炭素フローの決定のための手段としての使用のための消費者における脂質代謝経路の理解が欠かせません。念頭、 13C isotopologue のプロファイリングは、原理的にはすべての生物学的システムの17、炭素代謝の調査のため適用できる有望な方法であります。13C 標識炭素基板、 13C 代謝ネットワーク内の分布の導入は消費者を特定の isotopologue 分布の生成された代謝産物からトレースできます。これは定量核代謝共鳴分光法18,19または質量20,は21、その高いため低バイオマス後者の支持された生体試料によって評価されることができます。感度。
ただし、isotopologue プロファイルがされて正常にアミノ酸に適用し、細菌性病原体17,22,23, 脂肪酸の実装の体内の炭素代謝に洞察力を提供酸が遅れています。安定同位体標識前駆体脂肪酸、その食物ルーティングや劣化、土壌無脊椎動物の消費者に β 酸化を介しての運命の最初の詳細な分析、最近メンツェルらによって実行されました。24. 13C 標識脂肪酸 isotopologue による頻繁な土壌無脊椎動物、トビムシ、キーの子孫の後で導入実験の方法論的基礎を提供するここでは。これらの植栽は、土壌食物網とがよく、栄養マーカー脂肪酸8,25の調査の重要なコンポーネントは、フォームの良いモデル グループです。
栄養マーカー脂肪酸食物網生態学における定量的炭素フローの決定のための手段としての使用のための消費者における脂質代謝経路の理解が欠かせません。現在のプロトコルは、トビムシから実験、および抽出および支配的な脂質画分 (中性脂質、リン脂質) のメチル化のための生化学的なプロシージャを供給所のデザインと設定を与えます。質量分析法による脂肪酸の isotopologue 組成を分析する方法について説明し、関連の数式と計算について説明します。この手順の結果: (i) 1 つ以上によって (すなわち、脂肪酸分子イオン M+) 親イオンの質量を超える isotopologues の比質量単位 (M+1、+2M、M+3、等) および (ii) 全体1313C 標識前駆体由来の脂肪酸で C 濃縮。このアプローチは、これらは、成功したラベルの吸収量を確保するための制御された条件下で十分な量の培養とその後を前提に他の捕食者-被食者相互作用に適用できる一般にトビムシに使用、検証します。

<table><tbody><tr><td>neoLab-Round jars</td><td>neoLab</td><td>2-1506</td><td>69 x 40 mm, 10 pacs/pack</td></tr><tr><td>Charcoal activated</td><td>Carl Roth</td><td>X865.1</td><td>p.a., powder, CAS No. 7440-44-0</td></tr><tr><td>Alabaster Dental</td><td>R&amp;Ouml;HRICH-GIPSE</td><td>---</td><td>http://www.roehrich-gipse.de/dentalgipse.php</td></tr><tr><td>Chloroform</td><td>Carl Roth</td><td>7331.1</td><td>HPLC &amp;ge; 99,9 %</td></tr><tr><td>Methanol</td><td>Carl Roth</td><td>P717.1</td><td>HPLC &amp;ge; 99,9 %</td></tr><tr><td>Hexan</td><td>Carl Roth</td><td>7339.1</td><td>HPLC &amp;ge; 98 %</td></tr><tr><td>&lt;em&gt;tert&lt;/em&gt;-Butyl methyl ether (MTBE)</td><td>Carl Roth</td><td>T175.1</td><td>HPLC &amp;ge; 99,5 %</td></tr><tr><td>Aceton</td><td>Carl Roth</td><td>7328.2</td><td>HPLC &amp;ge; 99,9 %</td></tr><tr><td>NaOH</td><td>Carl Roth</td><td>6771.1</td><td>p.a. &amp;ge;99 %, in pellets</td></tr><tr><td>di-Natriumhydrogenphosphat</td><td>Carl Roth</td><td>P030.1</td><td>p.a. &amp;ge;99 % , water free</td></tr><tr><td>Na-dihydrogenphosphat Dihydrat</td><td>Carl Roth</td><td>T879.1</td><td>p.a. &amp;ge;99 %</td></tr><tr><td>Hypochloric acid (6 N)</td><td>VWR International</td><td>26,115,000</td><td>AVS TITRINORM vol. solution</td></tr><tr><td>Bond Elut (Columns)</td><td>Agilent Tech.</td><td>14102037</td><td>HF Bond Elut-SI, 500 mg, 3 mL, 50/PK</td></tr><tr><td>Pr&amp;auml;paratengl&amp;auml;ser Duran</td><td>Glasger&amp;auml;tebau Ochs</td><td>135215</td><td>&amp;Oslash; 16 x 100 mm, plus screw cap with handy knurl and integrated PTFE/silicone gasket</td></tr><tr><td>Supelco 37 Component FAME Mix</td><td>Sigma-Aldrich</td><td>47885-U Supelco</td><td>10&amp;nbsp;mg/mL in methylene chloride, analytical standard</td></tr><tr><td>FlowMesh</td><td>Carl Roth</td><td>2796.1</td><td>Polypropylene mesh, approximately 0.3 mm thick, with 1 mm strand spacing</td></tr><tr><td>Bacterial Acid Methyl Ester (BAME) Mix</td><td>Sigma-Aldrich</td><td>47080-U Supelco</td><td>10&amp;nbsp;mg/mL in methyl caproate, analytical standard</td></tr><tr><td>Methyl nonadecanoate</td><td>Sigma-Aldrich</td><td>74208</td><td>analytical standard &amp;ge; 98.0 %</td></tr><tr><td>Hexadecanoic acid-1-13C (Palmitic)</td><td>Larodan Fine Chemicals</td><td>78-1600</td><td>GC &amp;ge; 98.0 % (&lt;sup&gt;13&lt;/sup&gt;C: 99.0 %)</td></tr><tr><td>RVC 2-25 CDplus</td><td>Martin Christ Gefrier-trocknungsanlagen</td><td></td><td>Compact benchtop midi concentrator</td></tr><tr><td>Alpha 2-4 LDplus</td><td>Martin Christ Gefrier-trocknungsanlagen</td><td></td><td>Drying manifold</td></tr><tr><td>MZ 2C NT</td><td>Vacuubrand GMBH</td><td></td><td>Vacuum pump</td></tr><tr><td>Roto-Shake Genie</td><td>Scientific Industries</td><td></td><td>Combined rocking and rotating device</td></tr><tr><td>XP64 Micro Comparator</td><td>Mettler Toledo</td><td></td><td>Super high precision balance</td></tr><tr><td>GC-System 7890A</td><td>Agilent Tech.</td><td></td><td>Gas chromatograph</td></tr><tr><td>7000 GC/MS Triple Quad</td><td>Agilent Tech.</td><td></td><td>Triple Quad mass spectrometer</td></tr><tr><td>7683B Series Injector</td><td>Agilent Tech.</td><td></td><td>Sample injector</td></tr><tr><td>Heraeus Multifuge 3SR&lt;sup&gt;+&lt;/sup&gt;</td><td>Thermo Scientific</td><td></td><td>Centrifuge with 10 ml tube rotor</td></tr></tbody></table>

fatty acid 13c isotopologue profiling provides insight into trophic carbon transfer and lipid metabolism of invertebrate consumers

通常完全な分子として同化、マイナーまたはなしの変更は、異なる栄養段階の間食事ルーティングできるように消費者組織に転送されるため、脂肪酸 (FAs)、食物網の生態学的に有用なバイオ マーカーです。しかし、FA 栄養マーカー アプローチはまだ土壌動物相の脂質代謝の限られた知識によって妨げられます。この調査は、脂肪酸代謝経路の 2 つの広範な土壌トビムシ、 Protaphorura fimataとHeteromurus 光沢苔のトレーサーとして完全ラベル パルミチン酸 (13C16:0、99 の atom %) を使用しました。運命、この前駆体の新陳代謝の変更を調査するために isotopologue のプロファイリングの手法を提案する単一イオンのモニタリングを用いた質量分析法によって行います。さらに、上流の研究室実験を供給、抽出および支配的な脂質画分 (中性脂質、リン脂質) と関連数式および計算のメチル化して説明します。Isotopologue プロファイリング13C 標識前駆体由来の脂肪酸で全体の13C 濃縮収量のみではなく、また親イオン (すなわちFA の分子イオンの質量を超える isotopologues のパターンを生成M+) それぞれのラベルの 1 つまたは複数の質量単位(M+1、+2M、M+3等)で FA。この知識は、デノボ合成と比較して完全に消費された FA の栄養経路の比率に結論をことができます。Isotopologue のプロファイリングは、栄養相互作用を分離する土壌動物における脂肪酸代謝の評価のための便利なツールとして使用することをお勧めします。

トビムシの新鮮な重量と脂質含量 記述されている実験の過程で NLFAs および PLFAs の内容変わりませんでした大幅時間をかけて大幅24が、供試体の重量がわずかに増加したに対し。両方のパラメーターは、トビムシ標本の体力の良いレベルを示します。脂肪酸組成と同位体分析のためのサンプリング日に対応する実験中のトビムシの新鮮...

Watch this Scientific Journal Video about Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers at JoVE.com

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

脂肪酸13C Isotopologue プロファイリング栄養炭素転送および無脊椎動物消費者の脂質代謝への洞察力を提供します。

すなわち、分子全体消費者組織への伝達と脂肪酸の同化脂肪酸の栄養マーカー アプローチ変更が小さい土壌無脊椎動物の脂肪酸代謝の知識のギャップによって妨げられて無またはマイナー。Isotopologue ・ プロファイリングは、栄養相互作用を分離するための貴重なツールとして提供されます。

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

fatty-acid-13c-isotopologue-profiling-provides-insight-into-trophic

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

8.0K ビュー.  Humboldt-Universität zu Berlin. すなわち、分子全体消費者組織への伝達と脂肪酸の同化脂肪酸の栄養マーカー アプローチ変更が小さい土壌無脊椎動物の脂肪酸代謝の知識のギャップによって妨げられて無またはマイナー。Isotopologue ・ プロファイリングは、栄養相互作用を分離するための貴重なツールとして提供されます。

ビデオ: Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Profiling the Triacylglyceride Contents in Bat Integumentary Lipids by Preparative Thin Layer Chromatography and MALDI-TOF Mass Spectrometry

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune system that is protective against pathogenic microbes. Abnormal sebum production and chemical composition are also a clinical symptom of specific skin diseases. Sebum contains a complex mixture of lipids, including triacylglycerides, which is species-specific. The broad chemical properties exhibited by diverse lipid classes hinder the specific determination of sebum composition. Analytical techniques for lipids typically require chemical derivatizations that are labor-intensive and increase sample preparation costs. This paper describes how to extract lipids from mammalian integument, separate broad lipid classes by thin-layer chromatography, and profile the triacylglyceride contents using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This robust method enables a direct determination of the triacylglyceride profiles among species and individuals, and it can be readily applied to any taxonomic group of mammals.

Mammalian integument contains solvent-extractable lipids that can provide chemical compositions characteristic of individual species. This paper presents a routine method for separating broad lipid classes isolated from integumentary tissues using thin layer chromatography and determining the triacylglyceride profile by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

分取薄層クロマトグラフィーおよびMALDI-TOF質量分析によってバット外皮脂質中トリアシルグリセリドの内容をプロファイリング

The mammalian integument includes sebaceous glands that secrete an oily material onto the skin surface. Sebum production is part of the innate immune ...

化学

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

ターゲット安定同位体希釈液体クロマトグラフィー - 質量分析のための細胞の脂質の抽出

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

生物工学

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.
The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.

Phospholipid fatty acids provide information about the structure of soil microbial communities. We present methods for extraction from soil samples with a single-phase chloroform mixture, fractionation of extracted lipids using solid phase extraction columns, and methanolysis to produce fatty acid methyl esters, which are analyzed by capillary gas chromatography.

土壌中の微生物のリン脂質脂肪酸の抽出と分析

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about ...

環境学

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

魚油サプリメントの脂質プロファイルの迅速な評価のための強力なツールとしてNMR分光法

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

Determination of Total Lipid and Lipid Classes in Marine Samples

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being carbon- and hydrogen-rich they are also hydrophobic and can act as a solvent and absorption carrier for organic contaminants and thus can be drivers of pollutant bioaccumulation in marine ecosystems. Their hydrophobic nature facilitates their isolation from seawater or biological specimens: marine lipid analysis begins with sampling and then extraction in non-polar organic solvents, providing a convenient method for their separation from other substances in an aquatic matrix.
If seawater has been sampled, the first step usually involves separation into operationally defined &#39;dissolved&#39; and &#39;particulate&#39; factions by filtration. Samples are collected and lipids isolated from the sample matrix typically with chloroform for truly dissolved matter and colloids, and with mixtures of chloroform and methanol for solids and biological specimens. Such extracts may contain several classes from biogenic and anthropogenic sources. At this time, total lipids and lipid classes may be determined. Total lipid can be measured by summing individually determined lipid classes which customarily have been chromatographically separated. Thin-layer chromatography (TLC) with flame ionization detection (FID) is regularly used for the quantitative analysis of lipids from marine samples. TLC-FID furnishes synoptic lipid class information and, by summing classes, a total lipid measurement.
Lipid class information is especially useful when combined with measurements of individual components e.g., fatty acids and/or sterols, after their release from lipid extracts. The wide variety of lipid structures and functions means they are used broadly in ecological and biogeochemical research assessing ecosystem health and the degree of influence by anthropogenic impacts. They have been employed to measure substances of dietary value to marine fauna (e.g., aquafeeds and/or prey), and as an indicator of water quality (e.g., hydrocarbons).

This protocol is for the determination of lipids in seawater and biological specimens. Lipids in filtrates are extracted with chloroform or mixtures of chloroform and methanol in the case of solids. Lipid classes are measured by rod thin-layer chromatography with flame ionization detection and their sum gives the total lipid content.

海洋試料における総脂質および脂質クラスの決定

Lipids are largely composed of carbon and hydrogen and, therefore, provide a greater specific energy than other organic macromolecules in the sea. Being ...

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

開発と成人期における中間代謝フラックスの安定同位体プロファイル線虫（Caenorhabditis elegans）</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

生物学

安定同位体標識を用いた脂質構造のスクリーニング

Tracing de novo Lipids using Stable Isotope Labeling LC-TIMS-TOF MS/MS

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable isotope labeling (SIL) offers several advantages for the study of lipid distribution, mobilization, and metabolism, as well as de novo lipid synthesis. The successful implementation of the SIL technique requires the removal of interferences from endogenous molecules. In the present work, we describe a high-throughput analytical protocol for the screening of SIL lipids from biological samples; examples will be shown of lipid de novo identification during mosquito ovary development. The use of complementary liquid chromatography trapped ion mobility spectrometry and mass spectrometry allows for the separation and lipids assignment from a single sample in a single scan (&lt;1 h). The described approach takes advantage of recent developments in data-dependent acquisition and data-independent acquisition, using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation. The measurement of SIL at the fatty acid chain level reveals changes in lipid dynamics during the ovary development of mosquitoes. The lipids de novo structures are confidently assigned based on their retention time, mobility, and fragmentation pattern.

著者スポットライト:安定同位体標識を用いた複雑なリピドームサンプルの定量(英語)

Presented here is a method for screening lipid structures through stable isotope labeling by using their retention time, mobility, and fragmentation.

安定同位体標識 LC-TIMS-TOF MS/MS を用いた de novo 脂質の追跡

Lipids are highly diverse, and small changes in lipid structures and composition can have profound effects on critical biological functions. Stable ...

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes. 
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

スルー代謝経路の確認と発見 13</supタンパク質構成アミノ酸の> C標識

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

脂肪酸¹³C Isotopologue プロファイリング栄養炭素転送および無脊椎動物消費者の脂質代謝への洞察力を提供します。

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分取薄層クロマトグラフィーおよびMALDI-TOF質量分析によってバット外皮脂質中トリアシルグリセリドの内容をプロファイリング

ターゲット安定同位体希釈液体クロマトグラフィー - 質量分析のための細胞の脂質の抽出

土壌中の微生物のリン脂質脂肪酸の抽出と分析

魚油サプリメントの脂質プロファイルの迅速な評価のための強力なツールとしてNMR分光法

海洋試料における総脂質および脂質クラスの決定

開発と成人期における中間代謝フラックスの安定同位体プロファイル線虫（Caenorhabditis elegans）

安定同位体標識 LC-TIMS-TOF MS/MS を用いた de novo 脂質の追跡

安定同位体標識 LC-TIMS-TOF MS/MS を用いた de novo 脂質の追跡

安定同位体標識 LC-TIMS-TOF MS/MS を用いた de novo 脂質の追跡

スルー代謝経路の確認と発見^{13 C標識}

脂肪酸13C Isotopologue プロファイリング栄養炭素転送および無脊椎動物消費者の脂質代謝への洞察力を提供します。

要約

さらに動画を探す

分取薄層クロマトグラフィーおよびMALDI-TOF質量分析によってバット外皮脂質中トリアシルグリセリドの内容をプロファイリング

ターゲット安定同位体希釈液体クロマトグラフィー - 質量分析のための細胞の脂質の抽出

土壌中の微生物のリン脂質脂肪酸の抽出と分析

魚油サプリメントの脂質プロファイルの迅速な評価のための強力なツールとしてNMR分光法

海洋試料における総脂質および脂質クラスの決定

開発と成人期における中間代謝フラックスの安定同位体プロファイル線虫（Caenorhabditis elegans）

安定同位体標識 LC-TIMS-TOF MS/MS を用いた de novo 脂質の追跡

安定同位体標識 LC-TIMS-TOF MS/MS を用いた de novo 脂質の追跡

安定同位体標識 LC-TIMS-TOF MS/MS を用いた de novo 脂質の追跡

スルー代謝経路の確認と発見 13 C標識

脂肪酸¹³C Isotopologue プロファイリング栄養炭素転送および無脊椎動物消費者の脂質代謝への洞察力を提供します。

スルー代謝経路の確認と発見^{13 C標識}