עבודה זו נתמכה על ידי מזונות עבור Theme הבריאות דיסקברי באוניברסיטה אוהיו ואת מחלקת מזון למדע וטכנולוגיה באוניברסיטת אוהיו. המחברים מבקשים להודות מתקן NMR באוניברסיטה אוהיו ואת המתקן NMR באוניברסיטת פן סטייט.

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שינויים ואסטרטגיות לפתרון בעיות איכות ספקטרלי. Linewidth של אות NMR ובכך ההחלטה של ​​ספקטרום התמ&quot;ג תלוי מאוד shimming, שהוא תהליך של אופטימיזציה של הומוגניות של השדה המגנטי. עבור ניתוח שיגרתי, shimming 1D הוא נאות ו shimming 3D אינו נדרש, ב...

צריכת חומצות n -3 שומן בתזונה הוכיחה להיות מועיל נגד מספר תנאים כגון הפרעות לב 1, 2, 3, 4 מחלות דלקתיות וסכרת 5. התזונה המערבית נחשבת עניית n -3 חומצות שומן ולכן צריכת תוספי שמן דגים מומלצת לשפר את n -6 / n -3 איזון בתזונה של צרכן 1. למרות העלייה האחרונה צריך תוסף שמן דגים, נותרו שאלות בנוגע לבטיחות, אותנטיות, ואיכות של חלק מהמוצרים הללו. ניתוח ההלחנה המהיר ומדויק של תוספי שמן דגים הוא חיוני כדי להעריך את האיכות כראוי של מוצרים המסחריים אלה ולהבטיח בטיחות צרכן. השיטות הנפוצות ביותר להערכת תוספת של שמן דגיםים הם גז כרומטוגרפיה (GC) ו אינפרא אדום ספקטרוסקופיה (IR). בעוד אלה הם מאוד שיטות רגישות, הם סובלים ממספר חסרונות 6. ניתוח GC הוא זמן רב (4-8 שעות) כי הפרדת derivatization של תרכובות פרט נדרש 7 ו חמצון שומנים עלול להתרחש במהלך הניתוח 8, 9. בעוד ספקטרוסקופיה IR יכול להיות כמותי, מודל חיזוי נדרש לבנות באמצעות רגרסיה ריבועים לפחות חלקית (PLSR), אם כי יש יוצאים מן הכלל שבהם להקות IR ניתן לייחס יחיד במתחם 10. PLSR דורש ניתוח של מספר גדול של דגימות, אשר מגדיל את זמן הניתוח 11. מסיבה זו, יש עניין גובר בפיתוח מתודולוגיות אנליטיות חדשות המאפשרות ניתוח מדויק ומהיר של מספר גדול של דגימות שמן דגים. ארגונים כגון OffiCE של תוספי תזונה (ODS) במכונים הלאומיים לבריאות (NIH) לבין מינהל המזון והתרופות (FDA) שיתפו פעולה עם איגוד הרשמי אנליטית כימאים (AOAC) לפתח שיטות חדשות אלה 12, 13. אחת השיטות אנליטי המבטיחות ביותר עבור ההקרנה ואת ההערכה של מטריצות מרובות רכיבים, כגון תוספי תזונה, הוא תהודה מגנטית גרעינית (NMR) ספקטרוסקופיה 14, 15. יש NMR ספקטרוסקופיה מספר יתרונות: היא טכניקה שאינה הרסנית וכמותיים, זה דורש מינימאלי ללא הכנת מדגם, וזה מאופיין דיוק שחזור מצוין. בנוסף, ספקטרוסקופיה NMR היא מתודולוגיה ידידותית לסביבה משום שהיא מנצלת כמויות קטנות בלבד של ממסים. החיסרון העיקרי של ספקטרוסקופיה NMR הוא רגישות נמוכה יחסית לעומת analyti אחריםשיטות קאל, לעומת זאת, התקדמות טכנולוגית אחרונה מכשור כגון שדות מגנטיים חזקים, בדיקות קריוגני של קטרים ​​שונים, עיבוד נתונים מתקדם, ואת רצפים וטכניקות דופקים תכליתיים הגבירו את הרגישות עד בטווח ננומטר. בעוד מכשור NMR הוא עלות גבוהה, את החיים הארוכים של ספקטרומטרים תמ&quot;ג לבין יישומים הרבים של התמ&quot;ג להקטין את העלות של הניתוח בטווח הארוך. פרוטוקול וידאו מפורט זה נועד לעזור למתרגלים חדשים בתחום להימנע ממלכודות הקשורים 1 H ו- 13 ניתוח ספקטרוסקופיות C התמ&quot;ג של תוספי שמן דגים.

<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

התזונה המערבית היא עניה בחומצות n -3 שומן, ולכן צריכת תוספי שמן דגים מומלצת להגדיל את צריכת חומרים מזינות החיוניים אלה. מטרת עבודה זו היא להציג את הניתוח האיכותי וכמותי של תוספי שמן דגים כמוסים באמצעות ברזולוציה גבוהה 1 H ו- 13 ספקטרוסקופיה C NMR ניצול שני מכשירי תמ&quot;ג שונים; מגהרץ 500 ומכשיר 850 MHz. פרוטון שני (1 H) ופחמן (13 C) NMR ספקטרה יכולים לשמש לקביעת כמותית של המרכיבים העיקריים של תוספי שמן דגים. כימות של שומני תוספי שמן דגים מושגת באמצעות שילוב של אותות התמ&quot;ג המתאימים ספקטרום 1D הרלוונטי. תוצאות המתקבלות על ידי 1 H ו- 13 C תמ&quot;ג הן בהסכם טוב אחד עם השני, למרות שוני רזולוציה ורגישות בין שני גרעינים ואת שני המכשירים. הצעת 1 H NMRsa יותר לניתוח מהיר לעומת 13 C תמ&quot;ג, כמו הקשת יכולה להיות מוקלטת תוך פחות מ 1 דק &#39;, בניגוד 13 ניתוח C NMR, שנמשך מ 10 דקות עד שעה אחת. ספקטרום 13 C התמ&quot;ג, לעומת זאת, הוא הרבה יותר אינפורמטיבי. זה יכול לספק נתונים כמותיים על מספר גדול יותר של חומצות שומן בודדים יכולים לשמש לקביעת חלוקת מיקומית של חומצות שומן על עמוד השדרה גליצרול. גרעינים שניהם יכולים לספק מידע כמותי בלבד ניסוי אחד ללא צורך של צעדים טיהור או הפרדה. עוצמת השדה המגנטי משפיעה בעיקר ספקטרום 1 H NMR בשל ברזולוציה הנמוכה שלה בגין 13 C תמ&quot;ג, לעומת זאת, אפילו נמוכה עלות מכשירי תמ&quot;ג יכולים להיות מיושמים ביעילות כשיטה סטנדרטית בתעשיית המזון ומעבד איכות מלאה.

1 H ו- 13 ספקטרום C NMR נאספו עבור תוספי שמן דגים זמינים מסחרית באמצעות שני מכשירים NMR; MHz 850 ו ספקטרומטר 500 MHz. ספקטרה אלה יכולים לשמש לקביעת כמותי של הרכיבים של שמן דגים, כגון חומצה docosahexaenoic (DHA) וחומצה eicosapentaenoic (EPA), כמו גם תרכובות אחרות כגון n...

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

הנה, ברזולוציה גבוהה 1 H ו- 13 C גרעיני תהודה מגנטית (NMR) ספקטרוסקופיה שימש ככלי מהיר ואמין עבור ניתוח כמותי ואיכותי של תוספי שמן דגים כמוס.

NMR ספקטרוסקופיה כמו כלי חזק עבור ההערכה המהירה של פרופיל השומנים של תוספי שמן דגים

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

26.1K Views.  The Ohio State University.  הנה, ברזולוציה גבוהה 1 H ו- 13 C גרעיני תהודה מגנטית (NMR) ספקטרוסקופיה שימש ככלי מהיר ואמין עבור ניתוח כמותי ואיכותי של תוספי שמן דגים כמוס. 

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.

ניתוח התוכן של חומצות שומן והרכב בMicroalgae

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

Environment

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.

פשוט ומהיר פרוטוקול למדידת שומנים ניטרלי בתאי אצות באמצעות הקרינה

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

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.

יחיד-תפוקה משלימים שיטות אנליטיות ברזולוציה גבוהה עבור אפיון תערובות מורכבות חומר אורגני טבעי

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.

קביעת שיעורי השומנים והשומנים הכוללים בדגימות ימיות

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.

הערכה כמותית של Lipidome שמרים באמצעות Ionization electrospray ספקטרומטריית מסה

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

Biology

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).

ניתוח metabolomic של מוח חולדה על ידי ספקטרוסקופיה בתהודה מגנטית הגרעינית ברזולוציה גבוהה של תמציות רקמות

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

Neuroscience

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 Lipidomics של רקמות מכרסם

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

Biochemistry

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.

ריכוז מטבוליטים של צפיפות נמוכה עבור קהילות Planktonic metabolomics הסביבה באמצעות תהודה מגנטית גרעינית ספקטרוסקופיה

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.

ליפידומיקה ותעתיק במחלות נוירולוגיות

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.