The overall goal of this methodology is the detection of N-Acetylneuraminic acid and N-Glycolylneuraminic acid in the liver and milk of wild-type and CMAH knock-out mice using HPLC analysis. This method can help to answer questions in the field of glycoscience, specifically the effect genotypes of CMAH mouse models have on the actual sialic acid contents of these animals. The main advantage that this technique has in comparison to genetic screens is that relative sialic acid levels can be determined also for heterozygous mice.
Evaluating this method will be Cui Cao and Wang Wenjiao, two graduate students from our laboratory. Begin this procedure with collection of mouse milk and mouse liver tissue from CMAH knock-out mice as described in the text protocol. To isolate sialic acids from milk, transfer 50 microliters of milk into a 1.5 milliliter centrifuge tube, and add 1.2 milliliters of prepared aqueous acetic acid solution.
To isolate sialic acids from liver tissue, gently thaw 20 to 50 milligram of mouse liver and transfer it into a dounce tissue grinder. Add 1.2 milliliters of the aqueous acetic acid solution and homogenize the tissue by gentle shearing for 10 seconds. Transfer the resulting suspension into a 1.5 milliliter centrifuge tube.
Next, incubate the acidified milk or tissue suspension for four hours at 80 degrees Celsius. Then, centrifuge the sample at 14 thousand times g for 10 minutes. Transfer the top 1000 microliters of the supernatant into a fresh 1.5 milliliter centrifuge tube.
Remove the solvent by centrifugal evaporation at room temperature to complete dryness. Redissolve the sample in 500 microliters of distilled water. Next, prepare a microanion exchange column by transferring 200 milligrams of anion exchange resin per sample into empty 3 milliliter tubes with an attached stopcock.
Add 2 milliliters of the aqueous acetic acid solution to replace the chloride ions in the resin with acetate ion. Close the stopcock as soon as the solvent reaches the top of the resin. Gently add 2 milliliters of distilled water and open the stopcock.
Stop the flow as soon as most of the solvent reaches the top of the resin. Make sure that the resin is always covered with solvent. Then, wash the resin column two more times with two milliliters of distilled water for removing the excess acetate.
Transfer the resulting 500 microliters of sample solvent onto the resin column and discard the flow-through. Gently was the resin with 2 milliliters of distilled water. Elute the sample anions from the resin using 1 milliliter of a 50 millimolar ammonium acetate solution into a 1.5 milliliter centrifuge tube.
Remove the solvent by centrifugal evaporation at room temperature to dryness. Weigh between 5 and 8 milligrams of N-Acetylneuraminic acid on an analytical balance into a 1.5 milliliter centrifuge tube. Note the exact weight and add 164 microliters of distilled water for every milligram.
Obtain N-Glycolylneuraminic acid in smaller quantities at a moderate price, such as in 1 milligram aliquots. Add 154 microliters of distilled water directly to compound to obtain a stock solution of approximately 20 millimolar Neu5Gc. Transfer the solution into a 1.5 milliliter centrifuge tube.
This solution can be stored at minus 20 degrees Celsius for up to 12 months. Combine 5 microliters from each stock solution into a fresh 1.5 milliliter centrifuge tube. Then, remove the solvent by centrifugal evaporation at room temperature to dryness.
Add 20 microliters of the OPD solution to the sialic acid samples derived from mouse milk, mouse liver, or the mixed sialic acid standard. Vortex the sample vigorously for 30 seconds. Then, incubate the samples for four hours at 80 degrees Celsius in the dark.
After letting the samples cool down for five minutes, add 80 microliters of distilled water. Then, centrifuge the tubes at 14 thousand times g for one minute. Next, transfer 80 microliters from the supernatant into a 300 microliter high-recovery HPLC vial.
The derivitized sialic acid samples can be stored at four to six degrees Celsius for up to one week. Analyze the samples using a standard HPLC system connected to an online fluorescence detector. Use a reversed phase C18 column with a 250 millimeter length and 4.6 millimeter diameter for the analysis.
After preparing solvent A and solvent B, and setting up the HPLC run as detailed in the protocol, inject 50 microliters of the sample into the HPLC system. Monitor eluence using the fluorescence detector excitation wavelength of 373 nanometers and an emission wavelength of 448 nanometers. Finally, calculate the relative amount of Neu5Gc as described in the text protocol.
Shown here are representative HPLC chromatograms of fluorescence labeled sialic acids from milk samples, derived from homozygous knock-out CMAH mice and heterozygous knock-out CMAH mice. wild-type mice are also shown. Similarly, chromatograms of the mouse liver derived fluorescence labeled sialic acids are shown here.
Comparing the relative amounts of Neu5Gc shows that homozygous CMAH knock-out mice contain no Neu5Gc, whereas heterzygous CMAH knock-outs and wild-type mice contain between 10 and 75%Neu5Gc. Once mastered, samples can be prepared within eight hours if the procedure was performed properly. And the samples can be analyzed by HPLC autosampling overnight.
While attempting this procedure, it's important to remember to perform the isolation of sialic acid and the derivitization procedure on the same day. Following the procedure, other methods like mass spectrometric analysis can be performed in order to confirm the nature of the obtained fluorescent signals. The development of this technique allows researchers to explore the sialic acid distribution in biological samples.
Don't forget that working with concentrated acetic acid and formic acid can be extremely hazardous, and precautions such as wearing protective gear should always be taken while performing this procedure.
