Name: synthesis of a deuterated standard for the quantification of 2-arachidonoylglycerol in caenorhabditis elegans
Uploaded: 2019-09-21T14:00:00.000+00:00
Duration: 14 min 25 s
Description: Watch this Scientific Journal Video about Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans at JoVE.com

This work was supported by a research grant from the Agencia Nacional de Promoci&#243;n Cient&#237;fica y Tecnol&#243;gica (ANPCyT, PICT 2014-3693). J.F.d.L., G.P., and B.H.C. are fellows from CONICET. D.d.M. and G.R.L. are members of the Research Career of CONICET. We are thankful to Gonzalo Lamberto (INMET) for LC-MS/MS analysis and helpful discussion. Video shooting and editing has been done by Ramiro Ortega and Mar&#237;a Soledad Casasola from Direcci&#243;n de Comunicaci&#243;n de la Ciencia, Facultad de Ciencia Pol&#237;tica y Relaciones Internacionales, Universidad Nacional de Rosario in Rosario, Argentina.

1. 1-AG-d5 preparation
NOTE: For obtaining 1-AG-d5 as a deuterated internal standard for quantification assays, follow the protocol as detailed below.
<ol>
	<li>Differential protection
	<ol>
		<li>To only protect primary alcohols, first add 38 mg of glycerol-d8 to a 10 mL reaction tube using a Pasteur pipette and add a magnetic stirrer.</li>
		<li>Add 5 mL of anhydrous dichloromethane (DCM) using a 5 mL Hamilton syringe, and fill the tube with dry N2 to yield an inert atmosphere.</li>
		<li>Prepare a bath using a shallow Dewar flask filled with distilled ethyl acetate.</li>
		<li>Fit the hermetically closed reaction tube inside the bath and cool it by slowly adding liquid N2 to the ethyl acetate until the solvent is frozen. 
		CAUTION: Liquid violently boils at room temperature (RT) and can cause severe burns when contacting eyes and skin.</li>
		<li>Add 54 mg of anhydrous collidine using a Hamilton syringe. 
		CAUTION: Collidine is volatile and has a very strong and unpleasant scent.</li>
		<li>Add 70 mg of tert-butyldimethylsilyl chloride and stir the entire solution for 3 h at -78 &#176;C on a magnetic stirrer.</li>
		<li>After 3 h, leave the reaction to warm at RT and keep stirring for an additional 12 h.</li>
		<li>Add 2 mL of brine to quench the reaction.</li>
		<li>Extract the solution 3x with 2 mL of distilled dichloromethane using a separating funnel, saving the organic extract each time.</li>
		<li>Combine the three organic extracts and dry them over sodium sulfate.</li>
		<li>Evaporate the dichloromethane under reduced pressure in a vacuum rotary evaporator carefully to avoid solvent projections.</li>
		<li>Purify the crude mixture by column chromatography using silica gel as the stationary phase and a 10% increasing hexane/ethyl acetate gradient, starting from 100% hexane and finishing with 100% ethyl acetate.</li>
		<li>Combine the product-containing fractions and remove the solvent under reduced pressure in a vacuum rotary evaporator to obtain the pure 1-O,3-O-bis-(TBDMS) glycerol-d5 as a colorless liquid.</li>
	</ol>
	</li>
	<li>Esterification
	<ol>
		<li>Add 10 mg of the 1-O,3-O-bis(TBDMS)-glycerol-d5 (previously synthesized) to a 10 mL reaction tube using a Pasteur pipette and add a magnetic stirrer.</li>
		<li>Add 2 mL of anhydrous dichloromethane using a 5 mL Hamilton syringe, and fill the tube with dry N2 to yield an inert atmosphere.</li>
		<li>Cool the solution to 0 &#176;C using an ice bath.</li>
		<li>Add 36 mg of arachidonic acid using a multi-volume adjustable micropipette and stir.</li>
		<li>Add 15 mg of 4-dimethylaminopyridine and stir.</li>
		<li>Add 15 mg of N,N&#39;-diisopropylcarbodiimide using a multi-volume adjustable micropipette and stir.</li>
		<li>Let the mixture react at 0 &#176;C for 3 h.</li>
		<li>After 3 h, leave the reaction to warm at RT and keep stirring for an additional 12 h.</li>
		<li>Add 2 mL of water to quench the reaction.</li>
		<li>Extract the organic solution 3x with 2 mL of distilled dichloromethane (DCM) using a separating funnel.</li>
		<li>Place the three organic extracts in the same tube and dry them over sodium sulfate.</li>
		<li>Evaporate the dichloromethane under reduced pressure in a vacuum rotary evaporator carefully to avoid solvent projections.</li>
		<li>Purify the crude mixture by column chromatography using silica gel as the stationary phase and a 10% increasing hexane/ ethyl acetate gradient, starting from 100% hexane and finishing with 50% hexane/50% ethyl acetate.</li>
		<li>Combine the product-containing fractions and remove the solvent under reduced pressure in a vacuum rotary evaporator to obtain the pure 1-O, 3-O-bis(TBDMS)-2-AG-d5 as a yellowish liquid.</li>
	</ol>
	</li>
	<li>Deprotection
	<ol>
		<li>Add 15 mg of the 1-O,3-O-bis(TBDMS)-2-AG-d5 (previously synthesized) to a 10 mL reaction tube using a Pasteur pipette and add a magnetic stirrer.</li>
		<li>Add 2 mL of anhydrous THF using a 5 mL Hamilton syringe, and fill the tube with dry N2 to yield an inert atmosphere.</li>
		<li>Cool the solution to 0 &#176;C using an ice bath.</li>
		<li>Add 150 &#181;L dropwise of 1 M tetrabutylammonium fluoride solution in THF using a Hamilton syringe.</li>
		<li>Let the reaction warm to RT and stir for 1 h.</li>
		<li>After 1 h, add 2 mL of water to quench the reaction.</li>
		<li>Extract the solution 3x with 2 mL of distilled dichloromethane using a separating funnel.</li>
		<li>Combine the three organic extracts and dry them over sodium sulfate.</li>
		<li>Evaporate the dichloromethane under reduced pressure in a vacuum rotary evaporator to obtain the pure 1-AG-d5 as a yellowish liquid.</li>
	</ol>
	</li>
	<li>Monitor all reactions by thin layer chromatography performed on silica gel 60 F254 pre-coated aluminum sheets. Visualize the bands under a 254 nm UV lamp after staining with an ethanolic solution of 4-anisaldehyde.</li>
</ol>
2. Preparation of standard stock and measuring solutions
<ol>
	<li>Dissolve 1 mg of the internal standard 1-AG-d5 in 1 mL of ACN and sonicate for 1 min to obtain the 1,000 ppm standard stock solution.</li>
	<li>To prepare the 1,000 ppb solution used for quantification in worms, first prepare a 10 ppm solution: take 10 &#181;L of the stock solution using a Hamilton syringe and dilute it to a final volume of 1 mL by adding 990 &#181;L of ACN.</li>
	<li>Take 100 &#181;L from the solution produced in step 2.2 using a Hamilton syringe, and dilute it to a final volume of 1 mL by adding 900 &#181;L of ACN to obtain the 1,000 ppb solution used for the quantification.</li>
	<li>Sonicate for 1 min between each step to ensure complete solubilization. Store the solutions at -78 &#176;C to maintain the concentrations and integrity of the standards. After the standard solution is used, flow some nitrogen before closing the vial to prevent oxidation.</li>
</ol>
3. Growth and maintenance of C. elegans
NOTE: Seed the nematode growth medium (NGM) agar plates with E. coli OP50 and propagate the worms on these plates.
<ol>
	<li>Mix 3 g of NaCl with 17 g of agar.</li>
	<li>Add 2.5 g of peptone, then add 975 mL of H2O.</li>
	<li>Autoclave for 50 min, then cool the flask to 55 &#176;C.</li>
	<li>Mix the following: 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, 25 mL of 1 M KH2PO4 buffer (all of which have been previously autoclaved), and 1 mL of 5 mg/mL cholesterol in ethanol.</li>
	<li>While maintaining a sterile environment, dispense the NGM solution into 60 mm Petri plates, filling the plates to two-thirds of their volume. Store the plates at 4 &#176;C.</li>
	<li>Streak the E. coli bacterial culture from a -80 &#176;C glycerol stock onto the LB agar plate. Let it grow on the plate overnight at 37 &#176;C.</li>
	<li>Pick up a single colony to inoculate 100 mL of liquid LB overnight at 37 &#176;C with agitation. 
	NOTE: It is not necessary to check the O.D. because this strain can reach stationary phase over this time.</li>
	<li>Remove the stored NGM plates, remove the lids in the laminar flow hood, and leave open to allow evaporation of excess moisture from the plates.</li>
	<li>Once the plates are dried, use a Pasteur pipette to add 100 &#181;L of OP50 E. coli to the center of the plate without spreading.</li>
	<li>Leave the OP50 E. coli lawn to grow overnight at RT or at 37 &#176;C for 8 h.</li>
	<li>Add the desired number worm embryos obtained by hypochlorite treatment or &#34;bleaching&#34; (Section 4). 
	NOTE: Cool the plates to RT before the addition of worms.</li>
</ol>
4. Bleaching technique for synchronizing C elegans cultures
<ol>
	<li>Seed and chunk worms onto 6 cm NGM plates.</li>
	<li>Leave the worms growing for 2-3 days to obtain sufficient numbers of eggs and gravid adults on the plate.</li>
	<li>Once there are enough eggs/adults, pour 5 mL of M9 onto the plate.</li>
	<li>Transfer the worms to a 15 mL centrifuge tube using a glass pipette.</li>
	<li>Centrifuge the tube for 2 min at 2,000 x g and pellet the worms.</li>
	<li>Suction out most of the M9, avoiding disturbance of the worm pellet.</li>
	<li>Add 3 mL of bleaching solution (2:1:1 ratio of NaOH:NaOCl:H2O).</li>
	<li>Invert gently to mix the solution for 5 min or until the number of intact adult worms decreases. 
	CAUTION: Do not bleach for more than 5 min.</li>
	<li>Centrifuge for 1 min at 2,000 x g and suction most of the bleaching solution without disturbing the worm pellet.</li>
	<li>Add 15 mL of M9 and mix well.</li>
	<li>Centrifuge again at 2000 x g for 1 min.</li>
	<li>Suction out most of the M9 without disturbing the worm pellet.</li>
	<li>Repeat steps 4.10-4.12 one or two more times.</li>
	<li>Add 5 mL of fresh M9 and agitate.</li>
	<li>Let the eggs hatch overnight with gentle rocking.</li>
</ol>
5. Worm sample preparation
<ol>
	<li>Let the N2 embryos obtained by the bleaching procedure hatch overnight in M9 buffer (5 mL in a 15 mL centrifuge tube) at 20 &#176;C.</li>
	<li>Harvest the synchronized L1s by centrifuging the tube for 2 min at 2,000 x g.</li>
	<li>Wash the worms with M9 buffer 1x, then quantify the number of live L1 worms.</li>
	<li>Seed approximately 10,000 worms into NGM plates (10 cm diameter) with 1 mL of OP50 E. coli (previously dried).</li>
	<li>Incubate the plates for 48 h at 20 &#176;C until worms reach the L4 stage.</li>
	<li>Harvest the worms using cold M9 buffer in a 15 mL centrifuge tube, wash them 1x, then and transfer them to a 1.5 mL tube.</li>
	<li>Pellet the worms by centrifugation at 2,000 x g for 1 min, eliminate most of the supernatant, immerse the tubes in liquid nitrogen, and store at -80 &#176;C.</li>
</ol>
6. Lipid extraction
<ol>
	<li>Thaw approximately 100 &#181;L of frozen worm pellets belonging to N2 on ice, add 1.3 mL of methanol, and sonicate the sample for 4 min.</li>
	<li>Add 2.6 mL of chloroform, and 1.3 mL of 0.5 M KCl/0.08 M H3PO4 to a final ratio of 1:2:1, 1,000 ppb of the internal standard 1-AG-d5, and butylated hydroxytoluene as an antioxidant agent at a final concentration of 50 &#181;g/mL.</li>
	<li>Vortex the samples for 1 min and sonicate in an ultrasonic water bath for 15 min on ice.</li>
	<li>Vortex the samples 2x for 1 min and centrifuge for 10 min at 2,000 x g to induce the phase separation.</li>
	<li>Collect the lower phase and collect it in a clean tube, dry it under nitrogen, and resuspend the solid residue in 100 &#181;L of ACN.</li>
</ol>
7. Endocannabinoid analysis by HPLC-MS/MS
<ol>
	<li>Use liquid chromatography coupled with an ESI triple quadrupole mass spectrometer to detect and quantify 2-AG from nematode samples.</li>
	<li>Use the following ratio for reversed-phase HPLC: from 0.0-0.5 min H2O:ACN (40:60), 0.5-6.5 min H2O:ACN (40:60) to (25:75), 6.5-7.5 min H2O:ACN (25:75), 7.5-8.0 min H2O:ACN (25:75) to (40:60),&#160;8.0-12.0 min H2O:ACN (40:60).</li>
	<li>Maintain the column temperature at 40 &#176;C and set the autosampler tray temperature to 10 &#176;C.</li>
	<li>Set the following ionization conditions: positive-ion mode; drying gas (N2) temperature = 300 &#176;C; drying gas flow rate = 10 L/min; nebulizer pressure = 10 UA; and cap. voltage = 4 kV.</li>
	<li>For the analyte detection, use MRM with the following transitions: 379.2 m/z to 289.2 m/z for 2-AG; and 384.2 m/z to 289.2 m/z for 1-AG-d5.</li>
</ol>
8. Endocannabinoid quantification in worms
<ol>
	<li>Use deuterated internal standard 1-AG-d5 and calculate the peak area ratios of the analyte to the internal standard.</li>
	<li>Use the following transitions: 384.2 m/z to 287.2 m/z for 2-AG; and 379.2 m/z to 287.2 m/z for 1-AG-d5.</li>
	<li>Calculate the concentration of the endogenous 2-AG by comparing to the peak area ratios of the deuterated standard using the concentration value of the standard.</li>
</ol>

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The authors declare no conflicts of interest.

Endocannabinoids are a class of lipids that have been implicated in the regulation of dauer formation in C. elegans7. More specifically, the synthesis of polyunsaturated fatty acids (PUFAs) is important for cholesterol trafficking and the reproductive development of worms. It is revealed here that 2-AG, an arachidonic acid containing endocannabinoid, is responsible for restituting the dauer larva to its normal cycle in worms that have impaired cholesterol metabolism7</su...

Endocannabinoids regulate multiple biological processes in a variety of organisms and are conserved lipid mediators1. The first discovered and most well-characterized endocannabinoids are anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG). Endocannabinoids play many critical roles, including those involved in brain reward systems as well as drug addiction, memory, mood, and metabolic processes2. AEA and 2-AG are only synthesized when needed and have short life spans, and they are degraded through transport protein reuptake and hydrolysis3.
The use of animal models like Caenorhabditis elegans (C. elegans) has become important to study the large variety of biological processes including apoptosis, cell signaling, cell cycle, cell polarity, gene regulation, metabolism, ageing, and sex determination4,5. Additionally, C. elegans is an excellent model for studying the physiological roles of polyunsaturated fatty acids (PUFAs). AEA has been identified in C. elegans and is reduced under dietary restriction6. This deficiency extends the lifespan of the nematode through a dietary restriction mechanism that can be suppressed by supplementation with the endocannabinoid. Recently, it was discovered that 2-AG and AEA play fundamental roles in the regulation of cholesterol trafficking in C. elegans7. More importantly, it was determined that supplementation with exogenous 2-AG can rescue dauer arrest, which is caused by the impaired cholesterol trafficking in Niemann-Pick type C1 C. elegans mutants.
To gain a better understanding of 2-AG&#39;s relationship with cholesterol trafficking and other biological processes in the nematode (i.e., monoaminergic signaling, nociception and locomotion), it is crucial to study this endogenous metabolite and how it is affected under certain environmental and dietary conditions8,9,10,11,12,13. Therefore, it is imperative to design and optimize a method to detect and quantify endogenous 2-AG in C. elegans that is simple to use for scientists of different fields, especially those who study the nematode&#39;s behavior in relation to this endocannabinoid.
In 2008, Lethonen and coworkers succeeded in identifying 2-AG and AEA in C. elegans using LC-MS analytical methods14. In 2011, they managed to expand this technique to other endocannabinoids15. More recent work has shown other analytical methods that have been successful in detecting and quantifying endocannabinoids in C. elegans, including mass spectrometry and GC-MS16,17,18, and it has also been reported that similar analytical methods can be expanded to other models19.
Previously reported analytical methods used for quantifying 2-AG in biological samples usually involve the use of deuterated standards that are commercially acquired and require availability for the purchase20,21. Many analytical standards for LC-MS/MS quantification of endocannabinoids are commercially available from different providers. Nevertheless, they are expensive, are sensitive, and become oxidized over time, due to the presence of multiple double bonds. The most common versions of these standards are based on the octa-deuterated arachidonic acid and are suitable for quantification by isotope dilution LC-MS/MS14,22. Also, most of these standards are substituted in position 2 of the glycerol, making them unstable under most conditions since they are prone to acyl migration19,23.
To overcome the difficulties associated with the costs and stability of these deuterated standards, a convenient and simple method is presented to prepare an analytical standard based on glycerol-d5. The sequence to prepare the penta-deuterated standard requires a three-step procedure that results in the standard 1-AG-d5, which is stable and does not undergo acyl migration (the main issue when aiming to synthesize 2-monoacylglycerols).
The main objective here is to show a simple and reproducible method to study 2-AG in C. elegans, including the synthesis of the analytical deuterated standard, preparation and extraction of the nematode samples, and analysis by LC-MS/MS (Figure 1). This synthetic procedure is achievable without the sophisticated organic synthesis knowledge or special equipment, making it suitable for scientists from different fields who are studying C. elegans behavior under endocannabinoid influence. The method is also expandable to other study models, making it useful for different targets. The standard, prepared as reported here, has been applied to successfully develop a fast and reliable chromatographic method that allows for effective detection and quantification of 2-AG in a reproducible manner.

<table><tbody><tr><td>4-dimethylaminopyridine</td><td>Sigma-Aldrich</td><td>107700</td><td>reagent grade, 99%</td></tr><tr><td>antioxidant BHT</td><td>Sigma-Aldrich</td><td>W21805</td><td></td></tr><tr><td>Arachidonic acid</td><td>Sigma-Aldrich</td><td>10931</td><td></td></tr><tr><td>Glycerol-d&lt;sub&gt;8&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>447498</td><td></td></tr><tr><td>Mass detector Triple Quadrupole</td><td>Thermo Scientific</td><td></td><td>TSQ Quantum Access Max</td></tr><tr><td>N,N&amp;rsquo;-diisopropylcarbodiimide</td><td>Sigma-Aldrich</td><td>D125407</td><td></td></tr><tr><td>NMR spectrometer</td><td>Bruker</td><td></td><td>Avance II 300 MHz</td></tr><tr><td>reversed-phase HPLC column</td><td>Thermo Fisher</td><td>25003-052130</td><td>C18 Hypersil-GOLD (50 x 2.1 mm)</td></tr><tr><td>tert-Butyldimethylsilyl chloride</td><td>Sigma-Aldrich</td><td>190500</td><td>reagent grade, 97%</td></tr><tr><td>tetrabutylammonium fluoride</td><td>Sigma-Aldrich</td><td>216143</td><td>1.0M in THF</td></tr><tr><td>UHPLC System</td><td>Thermo Scientific</td><td></td><td>Ultimate 3000 RSLC Dionex</td></tr><tr><td>worm strain N2 Bristol</td><td>Caenorhabditis Genetics Center (CGC)</td><td></td><td></td></tr></tbody></table>

synthesis of a deuterated standard for the quantification of 2-arachidonoylglycerol in caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

An isotopically labeled analog was successfully synthesized from commercially available d8-glycerol and arachidonic acid using a 3-step synthetic method (Figure 2, Figure 3). These steps are straightforward and do not require sophisticated equipment, specially controlled conditions, or expensive reagents. Thus, this method is robust and may be successfully extended to synthesize monoacylglycerides containing different ...

Watch this Scientific Journal Video about Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans at JoVE.com

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

synthesis-deuterated-standard-for-quantification-2

Research

JoVE Journal

Biochemistry

7.2K Views.  Universidad Nacional de Rosario. Endocannabinoïden zijn geconserveerde lipidemedia mediators die meerdere biologische processen in een verscheidenheid van organismen reguleren. De eerste ontdekte endocannabinoïden zijn anandamide en 2-arachidonoylglycerol, 2-AG. De nematode C.elegans is een krachtig diermodel om een grote verscheidenheid aan biologische processen te bestuderen.Onlangs ontdekten we dat 2-AG een fundamentele rol speelt bij de regulering van de cholesterolhandel in C.elegans. Daarom werd het noodzakeli...

Video: Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

Neuroscience

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

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

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

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

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

Biology

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research.

Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

We present robust biochemical and microscopic methods for studying Caenorhabditis elegans lipid stores. A rapid, simple, fixing-staining procedure for fluorescent lipid droplet imaging leverages the spectral properties of the lipophilic dye Nile red. We then present biochemical measurement of triglycerides and phospholipids using solid phase extraction and gas chromatography-mass spectrometry.

The nematode C. elegans has emerged as an important  model for the study of conserved genetic pathways regulating fat metabolism as  it relates to human ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, and participate in various signaling pathways. Caenorhabditis elegans synthesizes all of the enzymes necessary to produce a range of omega-6 and omega-3 fatty acids. This, combined with the simple anatomy and range of available genetic tools, make it an attractive model to study fatty acid function. In order to investigate the genetic pathways that mediate the physiological effects of dietary fatty acids, we have developed a method to supplement the C. elegans diet with unsaturated fatty acids. Supplementation is an effective means to alter the fatty acid composition of worms and can also be used to rescue defects in fatty acid-deficient mutants. Our method uses nematode growth medium agar (NGM) supplemented with fatty acidsodium salts. The fatty acids in the supplemented plates become incorporated into the membranes of the bacterial food source, which is then taken up by the C. elegans that feed on the supplemented bacteria. We also describe a gas chromatography protocol to monitor the changes in fatty acid composition that occur in supplemented worms. This is an efficient way to supplement the diets of both large and small populations of C. elegans, allowing for a range of applications for this method.

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Caenorhabditis elegans is a useful model to explore the functions of polyunsaturated fatty acids in development and physiology. This protocol describes an efficient method of supplementing the C. elegans diet with polyunsaturated fatty acids.

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Worm-align and Worm_CP, Two Open-Source Pipelines for Straightening and Quantification of Fluorescence Image Data Obtained from Caenorhabditis elegans

An issue often encountered when acquiring image data from fixed or anesthetized C. elegans is that worms cross and cluster with their neighbors. This problem is aggravated with increasing density of worms and creates challenges for imaging and quantification. We developed a FIJI-based workflow, Worm-align, that can be used to generate single- or multi-channel montages of user-selected, straightened and aligned worms from raw image data of C. elegans. Worm-align is a simple and user-friendly workflow that does not require prior training of either the user or the analysis algorithm. Montages generated with Worm-align can aid the visual inspection of worms, their classification and representation. In addition, the output of Worm-align can be used for subsequent quantification of fluorescence intensity in single worms, either in FIJI directly, or in other image analysis software platforms. We demonstrate this by importing the Worm-align output into Worm_CP, a pipeline that uses the open-source CellProfiler software. CellProfiler&#8217;s flexibility enables the incorporation of additional modules for high-content screening.&#160;As a practical example, we have used the pipeline on two datasets: the first dataset are images of heat shock reporter worms that express green fluorescent protein (GFP) under the control of the promoter of a heat shock inducible gene hsp-70, and the second dataset are images obtained from fixed worms, stained for fat-stores with a fluorescent dye.

Worm-align and Worm_CP, Two Open-Source Pipelines for Straightening and Quantification of Fluorescence Image Data Obtained from Caenorhabditis elegans

Worm-align/Worm_CP is a simple FIJI/CellProfiler workflow that can be used to straighten and align Caenorhabditis elegans samples and to score whole-worm image-based assays without the need for prior training steps. We have applied Worm-align/Worm_CP to the quantification of&#160;heat-shock induced expression in live animals or lipid droplets&#160;in fixed samples.

An issue often encountered when acquiring image data from fixed or anesthetized C. elegans is that worms cross and cluster with their neighbors. This ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...

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.

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

Stimulated Raman scattering (SRS) microscopy allows selective, label-free imaging of specific chemical moieties and it has been effectively employed to image lipid molecules in vivo. Here, we provide a brief introduction to the principle of SRS microscopy and describe methods for its use in imaging lipid storage in Caenorhabditis elegans.

Lipid metabolism is a fundamental physiological process necessary for cellular and organism health. Dysregulation of lipid metabolism often elicits ...

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are crucial in constituting structural components of cell membranes, storing energy, and as signaling molecules. Regulation of lipid metabolism and signaling is essential to activate distinct longevity pathways. The roundworm Caenorhabditis elegans is an excellent and powerful organism to dissect the contribution of lipid metabolism and signaling in longevity regulation. Multiple research studies have described how diet supplementation of specific lipid molecules can extend C. elegans lifespan; however, minor differences in the supplementation conditions can cause reproducibility issues among scientists in different labs. Here, two detailed supplementation methods for C. elegans are reported employing lipid supplementation either with bacteria seeded on plates or bacterial suspension in liquid culture. Also provided herein are the details to perform lifespan assays with lifelong lipid supplementation and qRT-PCR analysis using a whole worm lysate or dissected tissues derived from a few worms. Using a combination of longitudinal studies and transcriptional investigations upon lipid supplementation, the feeding assays provide dependable approaches to dissect how lipids influence longevity and healthy aging. This methodology can also be adapted for various nutritional screening approaches to assess changes in a subset of transcripts using either a small number of dissected tissues or a few animals.

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

The present protocol describes lipid supplementation methods in liquid and on-plate cultures for Caenorhabditis elegans, coupled with longitudinal studies and gene transcriptional analysis from bulk or a few worms and worm tissues.

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

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