Este trabalho foi apoiado pelos Alimentos para Health Discovery tema no The Ohio State University e do Departamento de Ciência e Tecnologia de Alimentos no The Ohio State University. Os autores gostariam de agradecer a facilidade NMR no The Ohio State University ea facilidade NMR da Penn State University.

<ol>
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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. 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Modificações e Estratégias para a resolução de problemas Qualidade espectral. A largura de linha do sinal de NMR e, assim, a resolução do espectro de RMN é altamente dependente de calço, o qual é um processo para a optimização da homogeneidade do campo magnético. Para análises de rotina, 1D calçamento está adequada e uma shimming 3D não é necessária, uma vez que é realizada por pessoal de RMN em uma base regular. Se este não for o caso, um calço 3D deve se...

O consumo de n -3 ácidos gordos na dieta tem provado ser benéfico contra várias condições, tais como doenças do coração, 1, 2, 3, 4 doenças inflamatórias e diabetes 5. A dieta ocidental é considerada pobre em n -3 ácidos gordos e, portanto, o consumo de suplementos de óleo de peixe é recomendado para melhorar o n -6 / N -3 equilíbrio na alimentação 1 do consumidor. Apesar do recente aumento nos peixes consumo de suplemento de óleo, questões permanecem sobre a segurança, autenticidade e qualidade de alguns destes produtos. A análise de composição rápida e precisa de suplementos de óleo de peixe é essencial para avaliar adequadamente a qualidade destes produtos comerciais e garantir a segurança do consumidor. As metodologias mais comuns para a avaliação do suplemento de óleo de peixes são a cromatografia gasosa (GC) e espectroscopia de infravermelho (IR). Enquanto estes métodos são altamente sensíveis, eles sofrem de várias desvantagens 6. A análise por CG é demorado (4-8 h), porque a separação e derivatização de compostos individuais é necessária 7 e oxidação lipídica pode ocorrer durante a análise 8, 9. Enquanto a espectroscopia de IR pode ser quantitativa, é requerido um modelo de previsão a ser construído usando regressão de mínimos quadrados parciais (PLSR), apesar de haver excepções em que bandas de IR podem ser atribuídas a um único composto 10. PLSR requer a análise de um grande número de amostras, o que aumenta o tempo de análise 11. Por esta razão, existe um interesse crescente no desenvolvimento de novas metodologias analíticas que permitem a análise rápida e precisa de um grande número de amostras de óleo de peixe. Organizações, tais como o Office de suplementos dietéticos (ODS) dos Institutos Nacionais de Saúde (NIH) e da Food and Drug Administration (FDA) têm colaborado com a Associação de Químicos Analíticos Oficiais (AOAC) para desenvolver estes novos métodos 12, 13. Um dos métodos analíticos mais promissores para o rastreio e avaliação de matrizes multi-componentes, tais como suplementos dietéticos, é de Ressonância Magnética (RMN) nuclear 14, 15. espectroscopia de RMN tem várias vantagens: é uma técnica não-destrutiva e quantitativa, isto requer o mínimo de preparação da amostra, e caracteriza-se por uma excelente precisão e reprodutibilidade. Além disso, a espectroscopia de RMN é uma metodologia ambientalmente amigável porque utiliza apenas pequenas quantidades de solventes. O principal inconveniente da espectroscopia de RMN é a sua sensibilidade relativamente baixa em comparação com outros analytimétodos de cal, no entanto, avanços tecnológicos recentes em instrumentação, tais como campos magnéticos mais fortes, sondas criogénicos de vários diâmetros, processamento de dados avançados, e sequências de pulso versáteis e técnicas têm aumentado a sensibilidade até à gama de nM. Enquanto NMR instrumentação é de alto custo, a longa-vida de espectrómetros de RMN e as muitas aplicações da RMN reduzir o custo da análise a longo prazo. Este protocolo de vídeo detalhada destina-se a ajudar os novos profissionais no campo evitar perigos associados com um H e 13 C RMN análise espectroscópica de suplementos de óleo de peixe.

<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

A dieta ocidental é deficiente em n-3 ácidos gordos, por conseguinte, o consumo de suplementos de óleo de peixe é recomendado para aumentar a ingestão desses nutrientes essenciais. O objectivo deste trabalho é mostrar a análise qualitativa e quantitativa de suplementos de óleo de peixe encapsulados usando de alta resolução de 1 H e 13 C RMN utilizando espectroscopia de RMN dois instrumentos diferentes; um a 500 MHz e num instrumento de 850 MHz. Ambos protão (1 H) e de carbono (13 C) os espectros de RMN pode ser usado para a determinação quantitativa dos principais constituintes de suplementos de óleo de peixe. Quantificação dos lidos nos suplementos de óleo de peixe é conseguida através da integração dos sinais de RMN apropriados nos espectros de 1D relevante. Os resultados obtidos por 1 H e 13 C RMN estão em boa concordância com o outro, não obstante a diferença de resolução e sensibilidade entre os dois núcleos e os dois instrumentos. 1 H RMN ofertasa análise mais rápida em comparação com 13 C RMN, como o espectro pode ser gravado em menos de 1 min, em contraste com 13 C Análise de RMN, que tem a duração de 10 minutos a uma hora. O espectro de RMN de 13 C, no entanto, é muito mais informativa. Ele pode fornecer dados quantitativos para um maior número de ácidos gordos individuais e pode ser utilizado para determinar a distribuição posicionai de ácidos gordos no esqueleto de glicerol. Ambos os núcleos podem proporcionar informação quantitativa em apenas uma experiência sem a necessidade de purificação ou de separação de passos. A força do campo magnético afecta principalmente a espectros de RMN de 1 H, devido à sua baixa resolução em relação à RMN de 13 C, no entanto, os instrumentos de RMN ainda mais baixo custo pode ser eficazmente aplicado como um método padrão pelos laboratórios da indústria alimentar e de controlo de qualidade.

1 H e 13 C espectros de RMN foram recolhidos para suplementos de óleo de peixe, disponíveis comercialmente, utilizando dois instrumentos de RMN; um 850 MHz e um espectrómetro de 500 MHz. Estes espectros podem ser usadas para a determinação quantitativa de componentes de óleo de peixe, tais como o ácido docosahexaenóico (DHA) e ácido eicosapentaenóico (EPA), bem como outros compostos, tais como n -1 cadeias acilo e índice importante nutricionalmen...

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

Aqui, de alta resolução de 1 H e 13 C de Ressonância Magnética Nuclear (RMN) foi usada como uma ferramenta rápido e fiável para a análise quantitativa e qualitativa dos suplementos de óleo de peixe encapsulado.

Espectroscopia de RMN como uma ferramenta robusta para a avaliação rápida do perfil lipídico do óleo de peixes suplementa

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 ...

nmr-spectroscopy-as-robust-tool-for-rapid-evaluation-lipid-profile

Universidad de Cartagena UDEC

Research

JoVE Journal

Chemistry

26.0K visualizações.  The Ohio State University.  Aqui, de alta resolução de 1 H e 13 C de Ressonância Magnética Nuclear (RMN) foi usada como uma ferramenta rápido e fiável para a análise quantitativa e qualitativa dos suplementos de óleo de peixe encapsulado. 

Vídeo: 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.

Análise do conteúdo de ácidos graxos e composição em Microalgas

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

Ambiente

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.

Um simples e rápido protocolo para medir lipídios neutros em algas células usando fluorescência

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

Química

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.

Single-produção complementares técnicas analíticas de alta resolução para caracterização de misturas complexas de matéria orgânica Natural

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.

Determinação de Aulas De Lipídios e Lipídios Totais em Amostras Marinhas

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.

A avaliação quantitativa do Lipidome fermento usando Electrospray Espectrometria de Massa de Ionização

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).

Metabolômica Análise do cérebro de um rato de alta resolução Espectroscopia de Ressonância Magnética Nuclear de Extratos de Tecidos

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

Neurociência

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.

Shotgun Lipidômica de Tecidos de Roedores

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

Bioquímica

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.

Concentração de metabólitos de comunidades de baixa densidade fitoplanctônicas para Metabolomics ambiental utilizando Espectroscopia de Ressonância Magnética Nuclear

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.

Lipidomics e Transcriptomics em Doenças Neurológicas

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 ...

Label-Free Imaging of Lipid Storage Dynamics in Caenorhabditis elegans using Stimulated Raman Scattering Microscopy

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits obesity and many associated diseases including cardiovascular disorders, type II diabetes, and cancer. To advance the current understanding of lipid metabolic regulation, quantitative methods to precisely measure in vivo lipid storage levels in time and space have become increasingly important and useful. Traditional approaches to analyze lipid storage are semi-quantitative for microscopic assessment or lacking spatio-temporal information for biochemical measurement. Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology that enables rapid and quantitative detection of lipids in live cells with a subcellular resolution. As the contrast is exploited from intrinsic molecular vibrations, SRS microscopy also permits four-dimensional tracking of lipids in live animals. In the last decade, SRS microscopy has been widely used for small molecule imaging in biomedical research and overcome the major limitations of conventional fluorescent staining and lipid extraction methods. In the laboratory, we have combined SRS microscopy with the genetic and biochemical tools available to the powerful model organism, Caenorhabditis elegans, to investigate the distribution and heterogeneity of lipid droplets across different cells and tissues and ultimately to discover novel conserved signaling pathways that modulate lipid metabolism. Here, we present the working principles and the detailed setup of the SRS microscope and provide methods for its use in quantifying lipid storage at distinct developmental timepoints of wild-type and insulin signaling deficient mutant C. elegans.