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.

Sialic acids are a family of acidic monosaccharides often found on the termini of cell surface proteins or lipid glycoconjugates of higher animals. Herein we describe the enzymatic synthesis of the two isotopically labeled sialic acid derivatives d3-X-Gal-α-2,3-Neu5Ac and d3-X-Gal-α-2,3-Neu5Gc. Using deuterium oxide as the reaction solvent, deuterium atoms could be successfully introduced during the enzymatic epimerization and aldol addition reactions when the sialosides were generated. NMR and mass spectrometric analyses confirmed that the resulting sialosides were indeed tri-deuterated. These compounds may be of interest as internal standards in liquid chromatography/mass spectrometric assays for biochemical or clinical studies of sialic acids. This was further exemplified by the use of this tri-deuterated sialosides as internal standards for the quantification of sialic acids in meat and egg samples.