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

Summary

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

Abstract

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.

Introduction

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 hydroxylase^2,3. However, animal-based food products can be dietary sources of Neu5Gc^4,5,6, leading to the production of anti-Neu5Gc antibodies and therefore trigger an immune response towards Neu5Gc<....

Protocol

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

1. Use wild-type C57Bl/6 mice from the Comparative Medicine Centre of Yangzhou University (China).
   NOTE: Cmah knock-out mice were generated based on the information provided from previous Cmah knock-out studies

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals:
N-acetylneuraminic acid	Sigma	A0812
N-glycolylneuraminic acid	Sigma	50644	1 mg aliquot should be sufficient
o-Phenylenediamine	Sigma	694975
Sodium hydrogen sulfite	J&K Scientific Ltd	75234
Tools/Materials:
3 mL SPE tubes	Supelco	Sigma 57024	empty solid phase extraction columns
Luer stopcock	Sigma	S7396	to stop the flow of the SPE tube
Dowex 1X8	Dow Chemicals	Sigma 44340	200 - 400 mesh
Dounce tissue grinder	Sigma	D8938	tight fit
HPLC Analysis:
High-recovery HPLC vial	Agilent Technologies	#5188-2788
HPLC System	Shimadzu	Nexera
Fluorescence Detector for HPLC	Shimadzu	RF-20Axs
HPLC Column	Phenomenex	Hyperclone ODS	250 x 4.6 mm
LCMS-grade H2O	Merck Millipore	#WX00011
LCMS-grade Acetonitrile	Merck Millipore	#100029	Hypergrade
Ammonium hydroxide solution	Fluka	#44273	puriss. P.a.