The overall goal of this procedure is to analyze the fatty acid composition of liver. This is accomplished by first weighing out a sample of liver and homogenizing it. The second step is to extract all of the lipid from the homogenized liver sample.
Next, the individual lipid classes such as cholesterol esters are separated using solid phase extraction. The final step is ification and derivitization of the lipid classes into fatty acid methyl esters. Ultimately, gas chromatography is used to show how the fatty acid composition of tissues can change following nutritional intervention or be influenced by changes in physiological state such as pregnancy.
Although this method can provide an insight into tissue fatty acid composition, it can also be applied to other sample types such as oil, seeds, and foods. Generally, users new to this technique may struggle as some parts of the protocol require manual dexterity and need practice, such as the repetitive collection of upper and lower phases. Demonstrating the procedure will be Hena, Fisk and Annette West, a technician and PhD student from our laboratory.
First cut approximately 100 milligrams of frozen liver and weigh the tissue. Place the tissue into a plastic tube in an ice bucket and add 0.5 milliliters of ice cold, 0.9%sodium chloride. Then homogenize the tissue and transfer to a glass screw capped tube.
Use a further 0.3 milliliters of 0.9%sodium chloride to transfer any remaining tissue to the glass screw capped tube. Following this, add 100 micrograms of cholesterol heta dechen standard dissolved in two to one, dry chloroform to methanol to the tube, followed by an additional five milliliters of the same solvent solution containing 50 milligrams per liter of butylated hydroxy toluene. After adding one milliliter of one molar sodium chloride, cap the tube and mix the sample thoroughly by vortexing until it looks uniform.
Then centrifuge the sample at 1000 times G for 10 minutes with low break at room temperature. When finished, collect the lower phase using a glass past her pipette and transfer it to a new screw cap glass tube. Then dry the sample under nitrogen at 40 degrees Celsius.
At this point, connect the SPE tank to a vacuum pump and place an amino propyl silica SPE cartridge on the tank. Place a new screw cap glass tube labeled TAG and CCE in the tank rack under the column to collect the first fraction. Dissolve the total lipid extract in one milliliter of dry chloroform and mix the sample by vortexing.
Following this, transfer the sample to the column using a glass paste pipette and allow the liquid to drip into the screw cap tube under gravity. When the liquid no longer drips from the column, remove the remaining liquid by vacuum. Next, elute the TAG and CE fraction by washing the column twice with one milliliter of dry chloroform under vacuum.
When all of the liquid has alluded from the column, dry the TAG and CE fraction under nitrogen at 40 degrees Celsius. Place a new amino propyl silica SPE cartridge on the SPE tank and place a screw cap glass tube in the tank tray under the cartridge to collect the waste. Wash the column with one milliliter of dry hexane three times under vacuum.
After turning the vacuum off, wash the column with a final one milliliter of H hexane under gravity. After replacing the waste tube with a new screw cap glass tube labeled ce, dissolve the dried TAG and CE fraction in one milliliter of dry hexane and mix the sample by vortexing. Then transfer the sample to the column using a glass pass or pipette, and allow the liquid to drip through the column under gravity.
When the liquid no longer drips from the column, remove the remaining liquid under vacuum under vacuum. Wash the column twice with one milliliter of dry hexane to elute the CE and dry the collected fraction under nitrogen at 40 degrees Celsius. Following this, add 0.5 milliliters of dry toluene to the separated CE fraction and mix the sample by vortexing.
Add one milliliter of a previously prepared methylation reagent to the sample dissolved in dry toluene. Then cap the tube securely and mix Gently heat the sample for two hours at 50 degrees Celsius. Once the samples have cooled to room temperature, add one milliliter of neutralizing solution.
Next, add one of dry hexane to the sample After vortexing centrifuge the samples at 250 times G for two minutes with low break at room temperature. When finished, collect the upper fame phase and transfer it to a new non screw cap disposable glass tube. Dry the collected fame sample under nitrogen at 40 degrees Celsius.
After placing a waste tube into the SPE tank and a silica gel, SPE cartridge onto the tank, wash the column three times with one milliliter of dry hexane under vacuum and one milliliter of dry hexane under gravity. Following removal of the waste washes. Add a new waste tube into the tank, then dissolve the CE fame sample in one milliliter of dry hexane.
After mixing the sample by vortexing, transfer it to the column using a glass pass or pipette. Once the liquid has dripped through the column under gravity, wash the column three times with one milliliter of hexane under vacuum. Next, remove the waste washes and place a new nons screw cap tube labeled CE fame into the tank.
Elute the CE fame fraction with two one milliliters of 95 to five dry hexane to d ethyl ether. Then dry the collected fraction under nitrogen at 40 degrees Celsius, dissolve the dried fraction in 75 microliters of dry hexane, and mix the sample by vortexing after the sample has been mixed by vortexing. Transfer it to a GC auto sample vial after repeating the previous step.
Analyze the sample on a gas chromatograph if the protocol is followed successfully. A chromatogram with clear symmetrical, well-defined peaks and with minimal background noise should be obtained as illustrated here. If contamination has occurred, the chromatogram will show additional peaks and exhibit non-symmetrical or skewed peaks as shown here.
Contamination from free cholesterol will occur when running fame derived from CE unless the cholesterol is removed. The use of a prepared calibration mix allows for the identification of the fame in the sample. The calibration mix is run using the same instrument settings as the samples so that the chromatogram can be compared to the sample and peaks identified based on their retention times as illustrated here.
The 16 zero peak is highlighted here to illustrate the comparison of retention times from the calibration mix in the top chromatogram to the fatty acids. In the bottom sample trace, allowing correct labeling of fatty acids peak area is used to calculate the percentage of specific fatty acids within the total. Once data have been collected, it is useful to inspect them for outliers as shown here.
Outlier samples can then be further investigated and the extraction and or analysis repeated if necessary. As shown here, fame within rat liver CE are described as a percentage of total fatty acids within liver ce. These data describe the CE fatty acids in the liver of virgin or pregnant Rats fed for 20 days on one of three different diets.
Statistical analysis using two factor Innova reveals significant effects of diet and pregnancy upon the proportion of several fatty acids, as well as significant diet by pregnancy interactions. As an illustration, the onic acid content of liver CE in pregnant rats is more influenced by diet than that of virgin females. This technique allows for 20 samples per day to be completed from beginning to the end if it is performed correctly.
Once mastered, batching of samples can increase throughput and double the amount processed in a week. After watching this video, you should have a good understanding of how to analyze the fatty acid composition of individual lipid classes using solid phase extraction and gas chromatography. Don't forget that working with solvents can be extremely hazardous and precautions such as working in fume hoods and wearing suitable PPE should always be taken while performing this procedure.
