Quantitative Determination of De Novo Fatty Acid Synthesis in Brown Adipose Tissue Using Deuterium Oxide

Contrary to other metabolic pathways like glucose disposal, fatty acid synthesis is not routinely assessed, leading to incomplete interpretations of the metabolic status. The key benefits of this method is that it's relatively non-invasive, and it's likely a better reflection of fatty acid synthesis flux than using isotope-labeled glucose where changes in upstream glucose handling can impact results. Here, we focus on brown adipose tissue, but this method is potentially useful to study any tissue in any mouse model.

To begin, place the samples on dry ice. Take a pre-labeled microcentrifuge tube, place it on an analytical balance and tare the balance. Then place tweezers and a scalpel or steel razor blade on dry ice for 10 to 20 seconds to cool.

Use the tweezers to take the frozen tissue sample out of the tube and place it on a plastic weight boat, which can be positioned on a flat block of dry ice or another pre-cooled surface. Using the scalpel or steel razor blade, dissect a small portion of the tissue equivalent to 5 to 15 milligrams in weight, and place it in the microcentrifuge tube. Record the exact weight and repeat for each specimen.

Add one microliter per milligram of 10 millimolar hexadecanoic D31 acid, followed by three five millimeter stainless steel beads to each sample. Then add 250 microliters of methanol, 250 microliters of water, and 500 microliters of chloroform to each sample with beads. Place the tubes in a pre-cooled block of a grinding mill and mix the samples at a vibrational frequency of 25 hertz for five minutes.

Remove the beads using a magnet and centrifuge the samples at 12, 000 G for 10 minutes at four degrees Celsius. Using a micropipette, take a fixed volume of the bottom phase of each sample into correspondingly labeled microcentrifuge tubes. Add 500 microliters of chloroform to the remaining sample and repeat these steps.

Place the tubes with the combined lower phase under nitrogen gas or in a chloroform-resistant refrigerated centrifugal vacuum at four degrees Celsius until completely dry. Pipette 98 milliliters of anhydrous methanol into a glass media bottle. And slowly add two milliliters of anhydrous sulfuric acid in the fume hood to make 2%sulfuric acid in methanol.

Mix by swirling the closed bottle. Add 500 microliters of this 2%sulfuric acid in methanol solution to each sample and vortex briefly. Incubate the samples on the heat block at 50 degrees Celsius for two hours and add 100 microliters of saturated sodium chloride solution and 500 microliters of hexane to each sample.

Vortex the samples vigorously at room temperature for one minute and leave the samples to sit for one minute. The two phases should be apparent after this. Collect the upper phase into a fresh microcentrifuge tube.

Then repeat the addition of hexane. And after phase separation, collect the second upper phase samples into the same labeled tubes. Dry the samples at room temperature under nitrogen gas and resuspend the samples in 20 microliters of hexane per milligram of original tissue weight.

Transfer the samples immediately to a glass gas chromatography vial with a glass insert. Inject the samples into a single quadruple gas chromatography mass spectrometer to determine the abundance of fatty acid methyl esters or FAMEs isotopologues. Inject one microliter of sample into a split or splitless inlet at an inlet temperature of 270 degrees Celsius using helium as the carrier gas.

In labeled safe-lock microcentrifuge tubes, combine 10 microliters of each plasma sample or standard four microliters of 10 molar sodium hydroxide and four microliters of 5%acetone in acetonitrile. Perform this in triplicate for each sample. After incubating the samples overnight at room temperature, add 450 to 550 milligrams of sodium sulfate to each sample, add 600 microliters of chloroform to each tube in the fume hood, and vigorously vortex for 15 seconds.

Centrifuge the samples at 300 G for two minutes. Take labeled glass GCMS vials with glass inserts and transfer the triplicate and then 80 microliter aliquots of the supernatant from each sample to the vials. Cap the vials tightly for the GCMS analysis.

The percentage of D2O enrichment in the plasma of mice over multiple time points is shown here. It was found that body water is enriched in the range of 2.5 to 6%that a baseline level of deuterium enrichment in body water is rapidly achieved in one hour and maintained for the duration of the study. The mass isotopologue distribution of palmitate in brown adipose tissue after three days of D2O administration at room temperature and thermoneutrality is shown here.

This result shows a higher M1 and M2 deuterium enrichment at room temperature. Brown adipose tissue molar enrichment and plasma molar enrichment of a range of fatty acids after three days of D2O administration at room temperature and thermoneutrality are presented in this figure. The colder temperature enrichment was found in a broad range of fatty acids in brown adipose tissue.

Notably, plasma total fatty acid enrichment does not follow the same trend as brown adipose tissue, but instead, fatty acid enrichment is increased with thermoneutrality. These images represent the total abundance and the de novo synthesized palmitate in brown adipose tissue after three days of D2O administration. The result shows an increase in the total palmitate synthesis at room temperature.

Be careful when running fatty acids on the GCMS. Make sure palmitate is not overloaded as just can give artificially high isotope enrichment values. Additionally, when analyzing acetone on the GCMS, make sure there is no background acetone peak before running the samples, so run blanks to remove any acetone in the system.

This method looks at total fatty acids across multiple lipid classes, but if you want to understand how fatty acid synthesis is impacting specific lipid classes, you can first separate these lipid classes using chromatography and then extract fatty acids from these fractions. Because lipogenesis is a key contributor to normal development, but also to many diseases, this protocol can be useful in many fields.