Name: synthesis of a deuterated standard for the quantification of 2-arachidonoylglycerol in caenorhabditis elegans
Uploaded: 2019-09-21T14:00:00
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</su...

<ol>
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J., Komuniecki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoids+Stimulate+the+TRP+Channel-Dependent+Release+of+Both+Serotonin+and+Dopamine+to+Modulate+Behavior+in+C.+elegans.">Cannabinoids Stimulate the TRP Channel-Dependent Release of Both Serotonin and Dopamine to Modulate Behavior in C. elegans.</a> The Journal of Neuroscience. 39 (21), 4142-4152 (2019).</li><li>Batugedara, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helminth-Derived+Endocannabinoids+That+Have+Effects+on+Host+Immunity+Are+Generated+during+Infection.">Helminth-Derived Endocannabinoids That Have Effects on Host Immunity Are Generated during Infection.</a> Infection and Immunity. 86 (11), e00441 (2018).</li><li>Zhang, M. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+2-arachidonoylglycerol,+1-arachidonoylglycerol+and+arachidonic+acid+in+mouse+brain+tissue+using+liquid+chromatography/tandem+mass+spectrometry.">Simultaneous determination of 2-arachidonoylglycerol, 1-arachidonoylglycerol and arachidonic acid in mouse brain tissue using liquid chromatography/tandem mass spectrometry.</a> Journal of Mass Spectrometry. 45 (2), 167-177 (2010).</li><li>Zoerner, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+UPLC-MS/MS+quantification+of+the+endocannabinoids+2-arachidonoyl+glycerol+(2AG),+1-arachidonoyl+glycerol+(1AG),+and+anandamide+in+human+plasma:+Minimization+of+matrix-effects,+2AG/1AG+isomerization+and+degradation+by+toluene+solvent+extraction.">Simultaneous UPLC-MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: Minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction.</a> Journal of Chromatography B. 883-884, 161-171 (2012).</li><li>Ivanov, I., Borchert, P., Hinz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+simultaneous+determination+of+N-arachidonoylethanolamine,+N-oleoylethanolamine,+N-palmitoylethanolamine+and+2-arachidonoylglycerol+in+human+cells.">A simple method for simultaneous determination of N-arachidonoylethanolamine, N-oleoylethanolamine, N-palmitoylethanolamine and 2-arachidonoylglycerol in human cells.</a> Analytical and Bioanalytical Chemistry. 407 (6), 1781-1787 (2015).</li><li>Keereetaweep, J., Chapman, K. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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-d8</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&#8217;-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 ...

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