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

Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. The first discovered endocannabinoids are anandamide and 2-arachidonoylglycerol, 2-AG. The nematode C.elegans is a powerful animal model to study a large variety of biological processes.

Recently, we discovered that 2-AG plays a fundamental role in regulation of cholesterol trafficking in C.elegans. This is why it became imperative to design and optimize a method to study endogenous 2-AG by detection and quantification in C.elegans. Previously reported analytical methods to quantify 2-AG in biological samples usually involve the use of deuterated standards that are commercial acquired are required to have them available for purchase.

Nevertheless, they are expensive, and due to the presence of multiple double bonds, are sensitive to get oxidized over time. The most common version of the standards are based on the octadeuterated arachidonic acid being suitable for quantification by isotope dilution, by liquid chromatography, and posterior mass spectroscopy. To overcome the difficulties associated to the cost and stability of the deuterated standards to quantify 2-AG, we used a convenient and simple method to prepare an analytical standard based on deuterium-5-glycerol.

The sequence to prepare the pendeuterated standard requires a three steps procedure. Being possible to be kept in the protected form until it is needed. The main objective of this work is to show a complete method to study 2-AG in C.elegans from the synthesis of the analytical deuterated standard to the preparation and extraction of the nematode samples and finally, the analysis by liquid chromatography and mass spectroscopy.

For obtaining deuterated 1-AG as a deuterated internal standard for quantification assays follow a three step protocol. In order to only protect primary alcohols, first add 38 milligrams of glycerol deuterated to a 10 milliliter reaction tube using a Pasteur pipette and add a magnetic stirrer. Next, add five milliliter of anhydrous DCM using a five milliliters Hamilton syringe and fill the tube with dry nitrogen to give an inert atmosphere.

Prepare a bath using a shallow Dewar flask filled with distilled ethyl acetate. Fill the hermetically closed reaction tube inside the bath and cool it by slowly the adding liquid nitrogen to ethyl acetate until the solvent is frozen. Caution, liquid violently boils at room temperature and might cause severe burns if in contact with eyes and skin.

Next, add 54 milligrams of anhydrous collidine using a Hamilton syringe. Caution, collidine is volatile and has a very strong and unpleasant smell. At 70 milligrams of terbutile-dimethyl-cetylchloride and stir everything for three hours at minus 78 degrees on a magnetic stirrer.

Add two milliliters of brine to quench the reaction. Extract the solution with two milliliters of distilled dichloromethane three times using a separated funnel keeping the organic extracts each time. Then, combine the three organic extracts and dry over sodium sulfite.

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. For this esterification step, add 10 milligrams of the product previously stated to a 10 milliliter reaction tube using a Pasteur pippette and add a magnetic stirrer. Add two milliliter of anhydrous DCM using a five milliliters Hamilton syringe and fill the tube with dry nitrogen to give an inert atmosphere.

Cool the solution to zero degrees using an ice bath. Ad 36 milligrams of arachidonic acid using a multi-volume adjustable micropipette and stir. Then, add 15 milligrams of 40-methyl-aminopyridine and stir.

Add 15 milligrams of diacylpropylcarbodiimid using a multi-volume adjustable micropipette and stir. Add two milliliters of water to quench the reaction and then extract the solution with two milliliters of distilled DCM three times using a separating funnel. Combine the three organic extracts and dry over sodium sulfite, purify the crude mixture by column chromatography using silica gel as a stationary phase an then 10%increasing hexane ethyl acetate gradient starting from 100%hexane and finishing with 50 50%hexane ethyl acetate.

Finally, for the deprotection step, add 15 milligrams of the previously synthesized product to a 10 milliliters reaction tube using a Pasteur pipette and add a magnetic stirrer. Add two milliliters of anhydrous tetrahydrofuran using a five milliliters Hamilton syringe and fill the tube with dry nitrogen to give an inert atmosphere. Then cool the solution to zero degrees using an ice bath.

Add 150 microliters drop-wise of one molar solution of tetrabutylammonium fluoride in THF using a Hamilton syringe. After one hour, add two milliliters of water to quench the reaction. Extract the solution with two milliliters distilled DCM three times using a separating funnel.

Combine the three organic extracts and dry over sodium sulfite. For the bleaching procedure, turn worms onto seeded six centimeters NGM plates. Allow the worm to grow from two to three days so that there are lots of eggs and gravid adults on the plate.

Once you have plenty of eggs and adults, pour five milliliters of M9 onto the plate. Using a glass pipette, transfer the worms to a 15 milliliters falcon tube. Centrifuge the tube for two minutes at 2, 000 G and pellet the worms.

Suction most of the M9 without disturbing the worm pellet. Add three milliliters of bleaching solution and mix the solution gently by inverting for approximately five minutes or until you see a decrease in the number of intact adult worms. Once most of the bodies have dissolved, centrifuge at 2, 000 G for one minute.

Suction most of the bleaching solution without disturbing the worm pellet. Add 15 milliliters of M9 to the tube and mix well. Centrifuge again at 2, 000 G for one minute.

Suction most of the M9 without disturbing the worm pellet. For worm sample preparation, let N2 embryos obtained by bleaching procedure had in overnight in M9 buffer at 20 degrees. Then, harvest synchronized L1s by centrifuging the tube two minutes at 2, 000 G.Wash the worms with M9 buffer one more time and quantify the number of live L1.Seed approximately 10, 000 worms into NGM plates with one milliliter of OP 50 Escherichia coli previously dried.

Incubate the plates at least for 48 hours at 20 degrees until the worms reach the L4 stage. Harvest the worms using cold M9 buffer in a 50 milliliters falcon tube. Wash them one more time and transfer them into a 1.5 milliliters tube.

Pellet the worms by centrifugation at 2, 000 G for one minute. Eliminate most of the supernatant. Then, immerse the tube in liquid nitrogen and stir at minus 80 degrees.

Thaw approximately 100 microliters of frozen worm pellets belonging to N2 and then add 1.3 milliliters of methanol. After that, sonicate the sample for four minutes. After sonication, add 1, 000 ppb of the internal deuterated standard, 2.6 milliliters of chloroform, and 1.3 milliliters of 0.5 molar of potassium chloride, 0.08 molar of phosphoric acid to a final ratio of one to one.

Vortex the samples for one minute and then sonicate them in an ultrasonic water bath for 15 minutes on ice. Vortex the samples again twice for one minute and centrifuge them for 10 minutes at 2, 000 G to induce phase separation. Collect the lower phase into a clean glass tube, dry it under nitrogen, and resuspend it in 100 microliters of acetonitrile.

To achieve a successful quantification of the endogenous 2-AG, it was necessary to synthesize it's saturated analog using a three step method. Afterwards, it was added to worm samples, extracted and analyzed by high performance liquid chromatography, coupled to mass spectrometry. Used as an internal standard, the synthetic deuterated 1-AG was the tool to quantify the endogenous metabolite.

The method was optimized using the transitions for 2-AG and for deuterated 1-AG where glycerol molecules are lost. 2-AG endogenous from the C.elegans samples was successfully detected and quantified by isotopic dilution with the chemically synthesized deuterized 1-AG using high performance liquid chromatography coupled to mass spectrometry. Sample one was prepared with the deuterated standard 1-AG, while sample two without the standard.

Since the original concentration of deutrated standard in sample one and three was of 1, 000 ppb each, from the peak area ratio it is possible to calculate the endogenous concentration of 2-AG in the sample of 340 ppb for sample one and 360 ppb for sample three, giving an average of 350 ppb. The ratios were calculated as a quality between the peak areas of 2-AG and deuterated 1-AG, respectively, for two isolated samples. Both with the deuterated standard added previous to extraction.

Given the recently discovered importance of 2-AG in the enhancement of cholesterol trafficking and lack of information of how lipids influence that process, it was necessary a reliable detection method for this endocannabinoid. The successful development of a simple and robust synthetic method reported here to obtain deuterated analog 2-AG was a key step of this protocol. Most of the reported methods to quantify monoacylglycerols involve the use of commercially available analytical standards, which are usually expensive and unstable under regular storage conditions, making them a unreachable for lower budget laboratories.

However, this method solves this by proposing a synthesis of the standard using more accessible starting materials. The synthetic method is straight forward without sophisticated conditions needed, making it ideal to be performed in any laboratory with minimal equipment and access to reactants, even in a reduced budget. In summary, this new procedure describes a straight forward and reproducible way of detecting and quantifying 2-AG that will help to answer some of the questions raised after the description of the role of these endocannabinoids in cholesterol trafficking in C.elegans.