Name: nmr spectroscopy as a robust tool for the rapid evaluation of the lipid profile of fish oil supplements
Uploaded: 2017-05-01T11:45:00.000+00:00
Duration: 8 min 54 s
Description: Watch this Scientific Journal Video about NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements at JoVE.com

Questo lavoro è stato sostenuto dagli alimenti per la salute Discovery tema presso l&#39;Ohio State University e il Dipartimento di Scienze e Tecnologie Alimentari presso l&#39;Ohio State University. Gli autori desiderano ringraziare l&#39;impianto NMR presso l&#39;Ohio State University e l&#39;impianto NMR alla Penn State University.

<ol>
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Am Oil Chem Soc. 70, 225-228 (1993).</li><li>Igarashi, T., Aursand, M., Hirata, Y., Gribbestad, I. S., Wada, S., Nonaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nondestructive+quantitative+acid+and+n-3+fatty+acids+in+fish+oils+by+high-resolution+1H+nuclear+magnetic+resonance+spectroscopy.">Nondestructive quantitative acid and n-3 fatty acids in fish oils by high-resolution 1H nuclear magnetic resonance spectroscopy.</a> J. Am. Oil Chem. Soc. 77, 737-748 (2000).</li><li>Plans, M., Wenstrup, M., Saona, L. <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+Infrared+Spectroscopy+for+Characterization+Dietary+Omega-3+Oil+Supplements.">Application of Infrared Spectroscopy for Characterization Dietary Omega-3 Oil Supplements.</a> J. Am. Oil Chem. Soc. 92, 957-966 (2015).</li><li>Jian-hua, C. I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+Spectrum+Detection+of+Fish+Oil+DHA+Content+Based+on+Empirical+Mode+Decomposition+and+Independent+Component+Analysis.">Near-infrared Spectrum Detection of Fish Oil DHA Content Based on Empirical Mode Decomposition and Independent Component Analysis.</a> J Food Nutr Res. 2 (2), 62-68 (2014).</li><li>Millen, A. E., Dodd, K. W., Subar, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+vitamin,+mineral,+nonvitamin,+and+nonmineral+supplements+in+the+United+States:+The+1987,+1992,+and+2000+National+Health+Interview+Survey+results.">Use of vitamin, mineral, nonvitamin, and nonmineral supplements in the United States: The 1987, 1992, and 2000 National Health Interview Survey results.</a> J. of Am. Diet Assoc. 104 (6), 942-950 (2004).</li><li>Dwyer, J. T., 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=Progress+in+developing+analytical+and+label-based+dietary+supplement+databases+at+the+NIH+office+of+dietary+supplements.">Progress in developing analytical and label-based dietary supplement databases at the NIH office of dietary supplements.</a> J. Food Compos. Anal. 21, S83-S93 (2008).</li><li>Monakhova, Y. B., Ruge, I., Kuballa, T., Lerch, C., Lachenmeier, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+coenzyme+Q10+in+food+supplements+using+1H+NMR+spectroscopy.">Rapid determination of coenzyme Q10 in food supplements using 1H NMR spectroscopy.</a> Int. J. Vitam. Nutr. Res. 83 (1), 67-72 (2013).</li><li>Monakhova, Y. B., 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=Standardless+1H+NMR+determination+of+pharmacologically+active+substances+in+dietary+supplements+and+medicines+that+have+been+illegally+traded+over+the+internet.">Standardless 1H NMR determination of pharmacologically active substances in dietary supplements and medicines that have been illegally traded over the internet.</a> Drug Test. Anal. 5 (6), 400-411 (2013).</li><li>Berger, S., Braun, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 200 and more NMR experiments: a practical course. , (2004).</li><li>Knothe, G., Kenar, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+fatty+acid+profile+by+1H-NMRspectroscopy.">Determination of the fatty acid profile by 1H-NMRspectroscopy.</a> Eur. J. Lipid Sci. Technol. 106, 88-96 (2004).</li><li>Sacchi, R., Medina, J. I., Aubourg, S. P., Paolillo, I. G. L., Addeo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+High-Resolution+13C+NMR+Analysis+of+Lipids+Extracted+from+the+White+Muscle+of+Atlantic+Tuna+(Thunnus+alalunga).">Quantitative High-Resolution 13C NMR Analysis of Lipids Extracted from the White Muscle of Atlantic Tuna (Thunnus alalunga).</a> J. Agric. Food Chem. 41 (8), 1247-1253 (1993).</li><li>Dais, P., Misiak, M., Hatzakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+marine+dietary+supplements+using+NMR+spectroscopy.">Analysis of marine dietary supplements using NMR spectroscopy.</a> Anal. Methods. 7 (12), 5226-5238 (2015).</li><li>Pickova, J., Dutta, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+Oxidation+in+Some+Processed+Fish+Products.">Cholesterol Oxidation in Some Processed Fish Products.</a> J. Anal. Oil Chem. Soc. 80 (10), 993-996 (2003).</li><li>Siddiqui, N., Sim, J., Silwood, C. J. L., Toms, H., Iles, R. A., Grootveld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicomponent+analysis+of+encapsulated+marine+oil+supplements+using+high-resolution+1H+and+13C+NMR+techniques.">Multicomponent analysis of encapsulated marine oil supplements using high-resolution 1H and 13C NMR techniques.</a> J. of Lipid Rsrch. 44 (12), 2406-2427 (2003).</li><li>Sua&#180;rez, E. R., Mugford, P. F., Rolle, A. J., Burton, I. W., Walter, J. A., Kralovec, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13C-NMR+Regioisomeric+Analysis+of+EPA+and+DHA+in+Fish+Oil+Derived+Triacylglycerol+Concentrates.">13C-NMR Regioisomeric Analysis of EPA and DHA in Fish Oil Derived Triacylglycerol Concentrates.</a> J. Am. Oil Chem. Soc. 87, 1425-1433 (2010).</li><li>Youlin, X. A., Moran, S., Nikonowiczband, E. P., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Z-restored+spin-echo+13C+1D+spectrum+of+straight+baseline+free+of+hump,+dip+and+roll.">Z-restored spin-echo 13C 1D spectrum of straight baseline free of hump, dip and roll.</a> Magn. Reson. Chem. 46, 432-435 (2008).</li><li>Tengku-Rozaina, T. M., Birch, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positional+distribution+of+fatty+acids+on+hoki+and+tuna+oil+triglycerides+by+pancreatic+lipase+and+13C+NMR+analysis.">Positional distribution of fatty acids on hoki and tuna oil triglycerides by pancreatic lipase and 13C NMR analysis.</a> Eur. J. Lipid Sci. Technol. 116 (3), 272-281 (2014).</li><li>Berry, S. E. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+effects+of+dietary+fatty+acids+as+related+to+their+position+on+triglycerides.">Studies on effects of dietary fatty acids as related to their position on triglycerides.</a> Lipids. 36, 655-668 (2001).</li><li>Vlahov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regiospecific+analysis+of+natural+mixtures+of+triglycerides+using+quantitative+13C+nuclear+magnetic+resonance+of+acyl+chain+carbonyl+carbons.">Regiospecific analysis of natural mixtures of triglycerides using quantitative 13C nuclear magnetic resonance of acyl chain carbonyl carbons.</a> Magnetic Res. in Chem. 36, 359-362 (1998).</li></ol>

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Modifiche e strategie per la risoluzione Qualità spettrale. La larghezza di riga del segnale NMR e quindi la risoluzione dello spettro NMR è fortemente dipendente spessoramento, che è un processo per l&#39;ottimizzazione della omogeneità del campo magnetico. Per le analisi di routine, 1D spessoramento è adeguata e non è necessario uno spessoramento 3D, dato che essa è stata eseguita da personale NMR su base regolare. Se questo non è il caso, una spessoramento 3D deve es...

Il consumo di acidi grassi n -3 nella dieta ha dimostrato di essere utile contro diverse condizioni quali disturbi cardiaci 1, 2, 3, 4 e malattie infiammatorie diabete 5. La dieta occidentale è considerato povero di n -3 acidi grassi e quindi si raccomanda il consumo di integratori di olio di pesce per migliorare la n -6 / -3 n equilibrio nella nutrizione del consumatore 1. Nonostante il recente aumento del consumo di pesce supplemento di olio, le domande rimangono circa la sicurezza, l&#39;autenticità e la qualità di alcuni di questi prodotti. L&#39;analisi rapida ed accurata compositiva di supplementi di olio di pesce è essenziale per valutare correttamente la qualità di questi prodotti commerciali e garantire la sicurezza dei consumatori. Le metodologie più comuni per la valutazione del supplemento di olio di pesces sono gascromatografia (GC) e la spettroscopia a infrarossi (IR). Mentre questi sono metodi altamente sensibili, soffrono di diversi inconvenienti 6. Analisi GC richiede molto tempo (4-8 h) per la separazione e derivatizzazione dei singoli composti è richiesto 7 e l&#39;ossidazione dei lipidi possono verificarsi durante l&#39;analisi 8, 9. Mentre la spettroscopia IR può essere quantitativa, un modello di previsione è richiesta per essere costruito usando parziale minimi quadrati regressione (PLSR), anche se ci sono eccezioni in cui bande IR possono essere attribuiti ad un singolo composto 10. PLSR richiede l&#39;analisi di un gran numero di campioni, che aumenta il tempo di analisi 11. Per questo motivo, v&#39;è un crescente interesse per lo sviluppo di nuove metodologie analitiche che permettono l&#39;analisi precisa e veloce di un gran numero di campioni di olio di pesce. Organizzazioni come l&#39;Office di integratori alimentari (ODS) presso il National Institutes of Health (NIH) e la Food and Drug Administration (FDA) hanno collaborato con l&#39;Associazione dei chimici analitici ufficiali (AOAC) per sviluppare questi nuovi metodi 12, 13. Uno dei metodi analitici più promettenti per lo screening e la valutazione di matrici a più componenti, come integratori alimentari, è Nucleare spettroscopia a risonanza magnetica (RMN) 14, 15. spettroscopia NMR ha diversi vantaggi: è una tecnica non distruttiva e quantitativa, richiede minima o nessuna preparazione del campione, ed è caratterizzato da un&#39;eccellente precisione e riproducibilità. Inoltre, la spettroscopia NMR è una metodologia ecologico perché utilizza solo piccole quantità di solventi. Lo svantaggio principale della spettroscopia NMR è la sua sensibilità relativamente bassa rispetto ad altri analytiMetodi cal, tuttavia, i recenti progressi tecnologici nella strumentazione quali campi magnetici forti, sonde criogeniche di vari diametri, elaborazione dati avanzata e sequenze di impulsi versatili e tecniche hanno aumentato la sensibilità fino alla gamma nM. Mentre strumentazione NMR è alto costo, la lunga durata di spettrometri NMR e le molte applicazioni di NMR abbassare il costo delle analisi a lungo termine. Questo protocollo video dettagliato ha lo scopo di aiutare i nuovi operatori del settore evitare insidie associati con 1 H e 13 C NMR analisi spettroscopica di supplementi di olio di pesce.

<table><tbody><tr><td>Avance III 850 NMR instrument</td><td>Bruker</td><td></td><td></td></tr><tr><td>Avance III 500 NMR instrument</td><td>Bruker</td><td></td><td></td></tr><tr><td>TCI 5 mm probe</td><td>Bruker</td><td></td><td>Helium cooled inverse (proton deetected) NMR probe featuring three independent channels (&lt;sup&gt;1&lt;/sup&gt;H, &lt;sup&gt;13&lt;/sup&gt;C, &lt;sup&gt;15&lt;/sup&gt;N)</td></tr><tr><td>BBO prodigy 5 mm probe</td><td>Bruker</td><td></td><td>Nitrogen cooled observe (X-nuclei detected) probe, featuring two channels; one for &lt;sup&gt;1&lt;/sup&gt;H and &lt;sup&gt;19&lt;/sup&gt;F detectionand one for X-nuclei (covering from &lt;sup&gt;15&lt;/sup&gt;N to &lt;sup&gt;31&lt;/sup&gt;P)</td></tr><tr><td>Spinner turbin</td><td>Bruker</td><td></td><td>NMR spinners are made by polymer materials and they have a rubber o-ring to hold the NMR tube securely in place</td></tr><tr><td>Topspin 3.5</td><td>Bruker</td><td></td><td></td></tr><tr><td>deuterated chloroform</td><td>Sigma-Aldrich</td><td>&amp;nbsp;865-49-6</td><td>99.8 atom % D, contains 0.03 TMS</td></tr><tr><td>2,6-Di-&lt;em&gt;tert&lt;/em&gt;-butyl-4-methylphenol</td><td>Sigma-Aldrich</td><td>&amp;nbsp;128-37-0</td><td>purity &gt;99%</td></tr><tr><td>Fish oil samples</td><td></td><td></td><td></td></tr><tr><td>NMR tubes</td><td>New Era</td><td>NE-RG5-7</td><td>5mm OD Routine &amp;ldquo;R&amp;rdquo; Series NMR Sample Tube</td></tr><tr><td>BSMS</td><td>Bruker</td><td></td><td>Bruker Systems Management System; control system device</td></tr></tbody></table>

nmr spectroscopy as a robust tool for the rapid evaluation of the lipid profile of fish oil supplements

La dieta occidentale è povera di acidi grassi n -3, pertanto si raccomanda il consumo di supplementi di olio di pesce per aumentare l&#39;apporto di questi nutrienti essenziali. L&#39;obiettivo di questo lavoro è quello di dimostrare l&#39;analisi qualitativa e quantitativa di olio di pesce incapsulato utilizzando ad alta risoluzione 1 H e 13 C NMR utilizzando due diversi strumenti NMR; 500 MHz e uno strumento 850 MHz. Sia protone (1 H) e carbonio (13 C) spettri NMR possono essere utilizzati per la determinazione quantitativa dei principali costituenti degli integratori di olio di pesce. Quantificazione dei lipidi negli integratori di olio di pesce è ottenuta attraverso l&#39;integrazione dei segnali NMR appropriate nel relativi spettri 1D. Risultati ottenuti da 1 H e 13 C NMR sono in buon accordo con l&#39;altro, nonostante la differenza di risoluzione e sensibilità tra i due nuclei e due strumenti. Offerta 1H NMRsa un&#39;analisi più rapida rispetto a 13 C NMR, lo spettro può essere registrato in meno di 1 minuto, a differenza di analisi 13 C NMR, che dura da 10 minuti a un&#39;ora. Lo spettro NMR 13 C, tuttavia, è molto più informativo. Può fornire dati quantitativi per un maggior numero di singoli acidi grassi e può essere utilizzato per determinare la distribuzione posizionale degli acidi grassi sullo scheletro di glicerolo. Entrambi i nuclei possono fornire informazioni quantitative in un esperimento senza la necessità di purificazione o separazione gradini. La forza del campo magnetico colpisce soprattutto l&#39;spettri NMR 1 H a causa della sua bassa risoluzione rispetto a 13 C NMR, tuttavia, strumenti NMR costo ancora più basso possono essere efficacemente applicato come un metodo standard dai laboratori dell&#39;industria alimentare e controllo qualità.

1 H e 13 C NMR Gli spettri sono stati raccolti per olio di pesce integratori disponibili in commercio utilizzando due strumenti NMR; un 850 MHz e uno spettrometro a 500 MHz. Questi spettri possono essere utilizzati per la determinazione quantitativa di componenti di olio di pesce, come l&#39;acido docosaesaenoico (DHA) e acido eicosapentaenoico (EPA), come pure altri composti come n -1 catene acile e importante indice nutrizionalmente come il n -6 ...

Watch this Scientific Journal Video about NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements at JoVE.com

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

Qui, la spettroscopia ad alta risoluzione 1 H e 13 C Risonanza Magnetica Nucleare (NMR) è stato utilizzato come strumento di rapida e affidabile per l&#39;analisi quantitativa e qualitativa dei supplementi di olio di pesce incapsulati.

Spettroscopia NMR come uno strumento robusto per la rapida valutazione del profilo lipidico di olio di pesce integratori

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 ...

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25.9K Views.  The Ohio State University.  Qui, la spettroscopia ad alta risoluzione 1 H e 13 C Risonanza Magnetica Nucleare (NMR) è stato utilizzato come strumento di rapida e affidabile per l'analisi quantitativa e qualitativa dei supplementi di olio di pesce incapsulati. 

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

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

Analisi di acido grasso contenuto e la composizione in microalghe

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

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A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal fermentation processes and screening for new oil-rich strains requires a fast and reliable protocol for determination of intracellular lipid content. Current practices rely largely on gravimetric methods to determine oil content, techniques developed decades ago that are time consuming and require large sample volumes. In this paper, Nile Red, a fluorescent dye that has been used to identify the presence of lipid bodies in numerous types of organisms, is incorporated into a simple, fast, and reliable protocol for measuring the neutral lipid content of Auxenochlorella protothecoides, a green alga. The method uses ethanol, a relatively mild solvent, to permeabilize the cell membrane before staining and a 96 well micro-plate to increase sample capacity during fluorescence intensity measurements. It has been designed with the specific application of monitoring bioprocess performance. Previously dried samples or live samples from a growing culture can be used in the assay.

A simple protocol to determine the neutral lipid content of algal cells using a Nile Red staining procedure is described. This time-saving technique offers an alternative to traditional gravimetric-based lipid quantification protocols. It has been designed for the specific application of monitoring bioprocess performance.

Una semplice e rapida il protocollo per la misurazione lipidi neutri in cellule algali Uso fluorescenza

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Chimica

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

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

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

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

Acido grasso 13C Isotopologue profilatura fornisce approfondimenti trasferimento trofico carbonio e metabolismo dei lipidi dei consumatori invertebrati marini

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

Biochimica

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Singolo-velocità effettiva complementari ad alta risoluzione tecniche analitiche per la caratterizzazione di miscele complesse di sostanza organica naturale

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

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.

Determinazione delle classi lipidiche e lipidiche totali in campioni marini

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

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Una valutazione quantitativa del Lipidome lievito con ionizzazione Electrospray Spettrometria di massa

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Biologia

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Analisi Metabolomica di cervello di ratto da Alta Risoluzione Spettroscopia di Risonanza Magnetica Nucleare di estratti di tessuto

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Neuroscienze

Shotgun Lipidomics of Rodent Tissues

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have made lipidomics a powerful analytical tool for monitoring tissue lipidome compositions in homeostatic as well as disease states. This paper presents a step-by-step protocol for a shotgun lipid analysis method that supports the simultaneous detection and quantification of a few hundred molecular lipid species in different tissue and biofluid samples at high throughput. This method leverages automated nano-flow direct injection of a total lipid extract spiked with labeled internal standards without chromatographic separation into a high-resolution mass spectrometry instrument. Starting from sub-microgram amounts of rodent tissue, the MS analysis takes 10 min per sample and covers up to 400 lipids from 14 lipid classes in mouse lung tissue. The method presented here is well suited for studying disease mechanisms and identifying and quantifying biomarkers that indicate early toxicity or beneficial effects within rodent tissues.

Shotgun mass spectrometry-based lipidomics delivers a sensitive quantitative snapshot of a broad spectrum of lipid classes simultaneously in a single measurement from various rodent tissues.

Lipidomica shotgun di tessuti di roditori

Lipids play a vital role as essential components of all prokaryotic and eukaryotic cells. Constant technological improvements in mass spectrometry have ...

Concentration of Metabolites from Low-density Planktonic Communities for Environmental Metabolomics using Nuclear Magnetic Resonance Spectroscopy

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each other at the biochemical level1. Nuclear magnetic resonance (NMR) spectroscopy is one of several technologies, including gas chromatography&#x2013;mass spectrometry (GC-MS), with considerable promise for such studies. Advantages of NMR are that it is suitable for untargeted analyses, provides structural information and spectra can be queried in quantitative and statistical manners against recently available databases of individual metabolite spectra2,3. In addition, NMR spectral data can be combined with data from other omics levels (e.g. transcriptomics, genomics) to provide a more comprehensive understanding of the physiological responses of taxa to each other and the environment4,5,6. However, NMR is less sensitive than other metabolomic techniques, making it difficult to apply to natural microbial systems where sample populations can be low-density and metabolite concentrations low compared to metabolites from well-defined and readily extractable sources such as whole tissues, biofluids or cell-cultures. Consequently, the few direct environmental metabolomic studies of microbes performed to date have been limited to culture-based or easily defined high-density ecosystems such as host-symbiont systems, constructed co-cultures or manipulations of the gut environment where stable isotope labeling can be additionally used to enhance NMR signals7,8,9,10,11,12. Methods that facilitate the concentration and collection of environmental metabolites at concentrations suitable for NMR are lacking. Since recent attention has been given to the environmental metabolomics of organisms within the aquatic environment, where much of the energy and material flow is mediated by the planktonic community13,14, we have developed a method for the concentration and extraction of whole-community metabolites from planktonic microbial systems by filtration. Commercially available hydrophilic poly-1,1-difluoroethene (PVDF) filters are specially treated to completely remove extractables, which can otherwise appear as contaminants in subsequent analyses. These treated filters are then used to filter environmental or experimental samples of interest. Filters containing the wet sample material are lyophilized and aqueous-soluble metabolites are extracted directly for conventional NMR spectroscopy using a standardized potassium phosphate extraction buffer2. Data derived from these methods can be analyzed statistically to identify meaningful patterns, or integrated with other omics levels for comprehensive understanding of community and ecosystem function.

A method for metabolite extraction from microbial planktonic communities is presented. Whole community sampling is achieved by filtration onto specially prepared filters. After lyophilization, aqueous-soluble metabolites are extracted. This approach allows for application of environmental metabolomics to trans-omics investigations of natural or experimental microbial communities.

Concentrazione dei metaboliti a bassa densità Comunità planctoniche per Metabolomica ambientali utilizzando la spettroscopia a risonanza magnetica nucleare

Environmental metabolomics is an emerging field that is promoting new understanding in how organisms respond to and interact with the environment and each ...

Lipidomics and Transcriptomics in Neurological Diseases

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with various signaling or ligand function that can underscore the onset and progression of diseases. Often changing at the presymptomatic level, lipids are an emerging source of drug targets and biomarkers. Many neurological diseases exhibit neuroinflammation, neurodegeneration, and neuronal excitability as common hallmarks, partly modulated by specific lipid signaling systems. The interdependence and interrelation of synthesis of various lipids prompts a multilipid, multienzyme, and multireceptor analysis in order to derive the commonalities and specificities of neurological contexts and to expedite the unravelling of mechanistic aspects of disease onset and progression. Ascribing lipid roles to distinct brain regions advances the determination of lipid molecular phenotype and morphology associated with a neurological disease.
Presented here is a modular protocol suitable for the analysis of membrane lipids and downstream lipid signals along with mRNA of enzymes and mediators underlying their functionality, extracted from discrete brain regions that are relevant for a particular neurological disease and/or condition. To ensure accurate comparative lipidomic profiling, the workflows and operating criteria were optimized and standardized for: i) brain sampling and dissection of regions of interest, ii) co-extraction of multiple lipid signals and membrane lipids, iii) dual lipid/mRNA extraction, iv) quantification by liquid chromatography multiple reaction monitoring (LC/MRM), and v) standard mRNA profiling. This workflow is amenable for the low tissue amounts obtained by sampling of the functionally discrete brain subregions (i.e. by brain punching), thus preventing bias in multimolecular analysis due to tissue heterogeneity and/or animal variability. To reveal peripheral consequences of neurological diseases and establish translational molecular readouts of neurological disease states, peripheral organ sampling, processing, and their subsequent lipidomic analysis, as well as plasma lipidomics, are also pursued and described. The protocol is demonstrated on an acute epilepsy mouse model.

This article presents a modular protocol for tissue lipidomics and transcriptomics, and plasma lipidomics in neurological disease mouse models targeting lipids underlying inflammation and neuronal activity, membrane lipids, downstream messengers, and mRNA-encoding enzymes/receptors underlying lipid function. Sampling, sample processing, extraction, and quantification procedures are outlined.

Lipidomica e trascrittomica nelle malattie neurologiche

Lipids serve as the primary interface to brain insults or stimuli conducive to neurological diseases and are a reservoir for the synthesis of lipids with ...