Name: determination of sialic acids in liver and milk samples of wild-type and cmah knock-out mice.
Uploaded: 2017-07-14T15:00:00
Description: Watch this Scientific Journal Video about Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice. at JoVE.com

This work was supported in part by the Natural Science Foundation of China (grant numbers 31471703, A0201300537 and 31671854 to J.V. and L.L.), and the 100 Foreign Talents Plan (grant number JSB2014012 to J.V.).

Procedures involving animal subjects have been approved by the Ethical Committee of the Experimental Animal Center of Nanjing Agricultural University in accordance to the National Guidelines for Experimental Animal Welfare (Ministry of Science and Technology, PR of China, 2006) with the animals housed in a SPF facility (Permission ID: SYXK-J-2011-0037).
1. Cmah Knock-out Mouse Model
<ol>
	<li>Use wild-type C57Bl/6 mice from the Comparative Medicine Centre of Yangzhou University (Chi...

<ol>
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The herein presented protocol allows the phenotypical assessment of homozygous Cmah knock-out mice by analyzing and quantifying the relative amounts of Neu5Gc of milk and liver samples. The analysis was performed using a standard HPLC setup with fluorescence detection. The most critical step of this procedure is the preparation of the anion exchange columns and performing the anion exchange chromatography; to settle the resin properly and to collect the right washing and elution fractions takes a bit of practice...

N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the most common sialic acids in most mammals1. Although able of synthesizing Neu5Ac endogenously, humans are not capable of producing Neu5Gc due to a primary exon deletion on the CMAH gene encoding for a CMP-Neu5Ac hydroxylase2,3. However, animal-based food products can be dietary sources of Neu5Gc4,5,6, leading to the production of anti-Neu5Gc antibodies and therefore trigger an immune response towards Neu5Gc7. This dietary effect of Neu5Gc is suspected to contribute to chronic inflammation and various other diseases8,9,10. In order to comprehensively understand the effects of Neu5Gc in humans, an animal model for the systematic study of the effects of foodborne sialic acids is highly desirable. Although protocols based on polymerase chain reaction (PCR) for analyzing of knock-out mice are well established and a convenient way for the genotypical assessment, the functional analysis of phenotype on the metabolic level requires more specific analysis methods. The phenotype of a Cmah knock-out mouse model can be assessed by isolating and analyzing the composition of sialic acids in liver or milk samples. Several methods for the detection of sialic acids in animal tissues have been reported previously: reacting sialic acids with resorcinol11 or thiobarbituric acid12 result in the formation of a chromophoric product and can be simply analyzed using a platereader based setup, but only the total sialic acid content may be determined. Alternatively, the analysis of sialic acids was also described using gas chromatography13, MALDI-ToF mass spectrometry14 or amperometric methods15. However, the most commonly applied sialic acid analysis methods are based on hydrolytic release, followed by fluorescence derivatization and subsequent high performance liquid chromatography16,17,18.

<table border="0" cellpadding="0" cellspacing="0" width="684">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:147px;">Chemicals:</td>
			<td style="width:144px;"></td>
			<td style="width:143px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-acetylneuraminic acid</td>
			<td>Sigma</td>
			<td>A0812</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-glycolylneuraminic acid</td>
			<td>Sigma</td>
			<td align="right">50644</td>
			<td>1 mg aliquot should be sufficient</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">o-Phenylenediamine</td>
			<td>Sigma</td>
			<td align="right">694975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydrogen sulfite</td>
			<td>J&#38;K Scientific Ltd</td>
			<td align="right">75234</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tools/Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3 mL SPE tubes</td>
			<td>Supelco</td>
			<td>Sigma 57024</td>
			<td>empty solid phase extraction columns</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luer stopcock</td>
			<td>Sigma</td>
			<td>S7396</td>
			<td>to stop the flow of the SPE tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dowex 1X8</td>
			<td>Dow Chemicals</td>
			<td>Sigma 44340</td>
			<td>200-400 mesh</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dounce tissue grinder</td>
			<td>Sigma</td>
			<td>D8938</td>
			<td>tight fit</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Analysis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High-recovery HPLC vial</td>
			<td>Agilent Technologies</td>
			<td>#5188-2788</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC System</td>
			<td>Shimadzu</td>
			<td>Nexera</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Detector for HPLC</td>
			<td>Shimadzu</td>
			<td>RF-20Axs</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Column</td>
			<td>Phenomenex</td>
			<td>Hyperclone ODS</td>
			<td>250x4.6 mm</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">LCMS-grade H2O</td>
			<td>Merck Millipore</td>
			<td>#WX00011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Acetonitrile</td>
			<td>Merck Millipore</td>
			<td>#100029</td>
			<td>Hypergrade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium hydroxide solution</td>
			<td>Fluka</td>
			<td>#44273</td>
			<td>puriss. P.a.</td>
		</tr>
	</tbody>
</table>

determination of sialic acids in liver and milk samples of wild-type and cmah knock-out mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

A schematic overview of the described analysis method is shown in Figure 1 and includes the isolation of sialic acids from milk and liver samples of wild-type and Cmah knock-out mutant mice, and the fluorescence derivatization and HPLC analysis of these components. Figure 2 and Figure 3 show representative HPLC chromatograms of derivatized sialic acids of milk and liver samples from homo- an...

Watch this Scientific Journal Video about Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice. at JoVE.com

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

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