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

Summary

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).

Abstract

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'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.

Introduction

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....

Protocol

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.

1. Differential protection
   1. 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.
   2. 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.
   3. Prepare a bath using a shallow Dewar flask filled with distilled ethyl acetate.
   4. Fit the hermeti....

Results

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 .......

Discussion

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

Materials

Name	Company	Catalog Number	Comments
4-dimethylaminopyridine	Sigma-Aldrich	107700	reagent grade, 99%
antioxidant BHT	Sigma-Aldrich	W21805
Arachidonic acid	Sigma-Aldrich	10931
Glycerol-d8	Sigma-Aldrich	447498
Mass detector Triple Quadrupole	Thermo Scientific		TSQ Quantum Access Max
N,N’-diisopropylcarbodiimide	Sigma-Aldrich	D125407
NMR spectrometer	Bruker		Avance II 300 MHz
reversed-phase HPLC column	Thermo Fisher	25003-052130	C18 Hypersil-GOLD (50 x 2.1 mm)
tert-Butyldimethylsilyl chloride	Sigma-Aldrich	190500	reagent grade, 97%
tetrabutylammonium fluoride	Sigma-Aldrich	216143	1.0M in THF
UHPLC System	Thermo Scientific		Ultimate 3000 RSLC Dionex
worm strain N2 Bristol	Caenorhabditis Genetics Center (CGC)